Category Archives: Gene therapy

The spectrum of viral vectors is very broad including both delivery vehicles developed for transient short-term and permanent long-term expression. Moreover, the types of vectors are represented by both RNA and DNA viruses with either single-stranded (ss) or double-stranded (ds) genomes. The main groups of viral vectors applied for gene therapy are summarized below and in , followed by examples of both preclinical () and clinical findings (). Finally, the approval of viral vector-based drugs is discussed.

The most applied viral vectors are certainly based on adenoviruses [4]. Naked dsDNA adenoviruses possess a packaging capacity of 7.5 kb of foreign DNA providing short-term episomal expression of the gene of interest in a relatively broad range of host cells. The original adenovirus vectors generated strong immune responses, whereas the so-called gutted second and third generation vectors containing deletions have proven to elicit substantially reduced immunogenicity [5]. Much attention has been paid to engineering packaging cell lines for large scale production of recombinant particles of Good Manufacturing Practice (GMP)-grade to support clinical trials [6]. AAV vectors carry a small ssRNA genome, which allows packaging of only 4 kb inserts [7]. Generally, AAV is considered to generate low pathogenicity and toxicity and provides long-term transgene expression through chromosomal integration [8]. One limitation of using AAV relates to the immune response triggered by repeated administration [9]. This problem has been addressed by applying a different AAV serotype for each re-administration. Another issue relates to the limited packaging capacity of foreign DNA into recombinant AAV particles [10]. This shortcoming has been addressed by engineering dual AAV vectors [11].

Herpes simplex viruses (HSV) are large enveloped dsDNA viruses characteristic of their lytic and latent nature of infection, which result in life-long latent infection of neurons and allows for long-term transgene expression [12]. Deletion of HSV genes has generated expression vectors with low toxicity and an excellent packaging capacity of >30 kb foreign DNA [13]. In contrast to HSV, retroviruses possess a ssRNA genome with an envelope structure [14]. Typically, retroviruses are randomly integrated into the host genome, which has been problematic, as previously described, in the therapy of SCID patients [2,3]. However, this shortcoming has triggered the development of safer vectors showing targeted integration and also improved helper cell lines [15]. Retroviruses can accommodate up to 8 kb of foreign inserts and have represented the gold standard vectors for long-term gene therapy applications. One drawback of retroviruses is their incapability to infect nondividing cells, which has enhanced the interest in application of lentivirus vectors for gene therapy. Although lentiviruses belong to the family of retroviruses, they have the capability of infecting both dividing and nondividing cells providing low cytotoxicity [16,17]. Possessing the same packaging capacity and chromosomal integration as conventional retroviruses, lentiviruses have become attractive for therapeutic applications requiring long-term expression.

Self-amplifying ssRNA viruses comprise of alphaviruses (Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis virus, and M1) and flaviviruses (Kunjin virus, West Nile virus, and Dengue virus) possessing a genome of positive polarity [18]. In contrast, rhabdoviruses (rabies and vesicular stomatitis virus) and measles viruses carry negative strand genomes [18]. Most of the self-amplifying RNA viruses possess a packaging capacity of 68 kb, and generate high levels of short-term transient gene expression [19]. Additionally, the ssRNA paramyxovirus Newcastle disease virus (NDV) replicates specifically in tumor cells and has therefore been frequently applied for cancer gene therapy [20]. Moreover, oncolytic cancer cell targeting vectors have been engineered for many of the listed ssRNA viruses above [21]. Another family of nonenveloped ssRNA viruses, namely Coxsackieviruses belonging to Picornaviridae, have been applied as oncolytic vectors [22,23].

Also, poxviruses and especially vaccinia viruses have been applied as delivery vectors [24]. The characteristic feature of poxviruses is their dsDNA genome, which can generously accommodate more than 30 kb of foreign DNA. Tumor-selective replication-competent poxvirus vectors have been engineered causing necrosis in nonhuman primates [25]. Additionally, vaccinia vectors, which replicate in tumor cells without damaging normal cells, were engineered by deletions in the thymidine kinase (TK) and vaccinia growth factor (VGF) genes [26].

Due to the many gene therapy applications of a number of viral vectors evaluated in preclinical animal models, only some examples can be presented here (). In this context, oncolytic adenoviruses have shown great promise in cancer therapy [27]. For instance, an oncolytic adenovirus engineered with a pancreatic cancer-targeting ligand SYENFSA (SYE), specifically infected and replicated in cancer cells, but not normal cells, provided effective oncolysis of pancreatic ductal adenocarcinoma PDAC) [28]. The AdSur-SYE vector, regulated by the survivin promoter, also showed high transduction efficiency in pancreatic neuroendocrine tumors (PNETs) [29]. Intratumoral administration of AdSur-SYE resulted in complete regression of subcutaneous tumors in a mouse model. In another approach, chimeric Adenovirus type 5 and type 3 vectors, which can selectively replicate in cancer cells, have been engineered [30]. Providing simultaneous expression of the secreted melanoma differentiation associated gene-7 (MDA-7) and interleukin-12 (L-24) from the chimeric Ad5/3 vector generated selective tumor cell death after intratumoral injection in animal models. Moreover, therapeutic activity was also confined to noninfected distant tumors due to the so-called bystander anti-tumor activity. To further enhance the therapeutic efficacy, the chimeric Ad5/3 vector was encapsulated in microbubbles for stealth delivery. Ultrasound treatment released and allowed replication of the vector, which together with secretion of MDA-7/IL-24 enhanced therapeutic activity, including promotion of apoptosis and inhibition of tumor angiogenesis. Due to the generally limited duration of therapeutic activity of adenovirus-based gene therapy, hybrid adenovirus vectors utilizing the Sleeping Beauty transposase system or clustered regularly interspaced short palindromic repeats (CRISPR) associated protein-9 nuclease have been used for chromosomal integration and permanent gene editing, respectively [31]. Oncolytic adenovirus vectors have also been used in combination with the expression of immunomodulatory proteins [32]. This approach can change the tumor microenvironment from immune-suppressive to immune-vulnerable due to activation of cytotoxic T cells. In another approach, the oncolytic adenovirus Enadenotucirev, an Ad11p and Ad3 chimeric vector, has demonstrated selective propagation and killing of tumor cells [33]. Due to the inability of replication in animal cells, Enadenotucirev was evaluated in a panel of primary human cells, which demonstrated >100-fold higher viral genome levels in tumor cells than in normal cells [33]. Furthermore, intravenous tolerability was assessed in mice. The resistance to inactivation by human blood components will potentially enable intravenous vector administration.

The X chromosome-linked neurodevelopmental disorder named Rett Syndrome (RTT) has been targeted by AAV vectors in a mouse model for RTT [34]. AAV vectors expressing the transcription regulator methyl CpG-binding protein 2 (MeCP2) delivered directly to the cerebrospinal fluid (CSF), showed dose-dependent side effects, but also extended survival of RTT mice. Moreover, the fatal neurodegenerative Huntingtons disease (HD) has been evaluated for AAV-based therapy in a HD mouse model [35]. Transgenic HD sheep expressing the full-length human huntingtin (HTT) gene were injected with AAV9 miRNA targeting exon 48 of the human HTT mRNA. The outcome was reduced human HTT mRNA and 5080% HTT protein in the striatum, indicating safe and effective gene silencing. Cystic fibrosis has been targeted by AAV-based expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in a number of animal models showing a good safety profile, although no clear clinical benefits [36]. Recently, the AAV1 and AAV5 serotypes were tested using a dual-luciferase reporter system based on firefly and Renilla luciferases, respectively [36]. Both AAV1 and AAV5 were delivered into lungs of Rhesus macaques by microspraying, which resulted in a 10-fold higher vector genome number of AAV1 than AAV5. However, the AAV1-based luciferase activity was not statistically higher in comparison to AAV5. Moreover, serum neutralizing antibodies showed a dramatic increase for both AAV serotypes. There were no adverse events, indicating safe administration of AAV, which supports additional clinical trials, especially with the more lung-tropic AAV1 serotype. In another approach, AAV vectors have been applied for the treatment of Duchenne and limb girdle muscular dystrophies [37]. Furthermore, dual AAV technology allowed the expression of a 7 kb canine H2-R15 mini-dystrophin gene using a pair of dual AAV vectors [38]. The AAV9 was administered to the extensor carpi ulnaris muscle in a canine model for Duchenne muscular dystrophy. The outcome was widespread mini-dystrophin expression, restoration of dystrophin-associated glycoprotein complex, reduced muscle degeneration, and improved myofiber size distribution. In the context of hemophilia A, liver-specific promoter and enhancer elements with a codon-optimized human coagulation factor VIII (hFVIII) gene have been engineered [39]. One promoter-enhancer construct with high hFVIII immunogenicity was evaluated in an FVIII knockout mouse model applying AAV8, AAV9, AAVhu37, and AAVrh64R1 vectors. Based on the generation of anti-hFVIII antibodies, the vectors were divided into one group, where less than 20% of mice (AAV8 and AAV9) and the other with more than 20% of mice (AAVrh10, AAVhu37 and AAVrh64R1) generated anti-hFVIII antibodies.

Due to the long-term effect, HSV vectors have found many applications in various disease areas. For instance, HSV-based expression of proinflammatory cytokines has proven useful in treatment of painful diabetic neuropathy [40]. In this context, continuous delivery of HSV-IL-10 into the nerve fibers of mice with type I diabetes blocked nociceptive and stress responses in transduction of the dorsal root ganglion (DRG) [40]. It was suggested that macrophage activation in the peripheral nervous system is involved in the pathogenesis of pain and that HSV-based cytokine expression inhibited the development of painful neuropathy. In another approach, administration of nonreplicating HSV vectors expressing growth factors in the skin of mice resulted in the transduction of DRGs and prevented the progression of sensory neuropathy without causing any side effects [41]. Related to cancer, oncolytic HSV vectors have been applied in several preclinical studies [42]. The genome of the HSV-1 HF10 vector includes nonengineered deletions and mutations and frame-shift mutations lacking the expression of UL43, UL48.5, UL55, UL56, and latency-associated transcripts, while demonstrating overexpression of UL53 and UL54. HSV-1 HF10 replicates efficiently in tumor cells causing extensive cytotoxic damage. Moreover, activated CD4+ and CD8+ T cells and natural tumor killer cells were induced in tumors resulting in significant tumor growth reduction and prolonged survival. Oncolytic HSV-2 vectors have also been evaluated in animal studies on colon cancer cells and cancer stem-like cells (CSLCs) and are known to be tumorigenic and responsible for cancer recurrence and metastasis [43]. Significant inhibition of tumor growth was observed after administration of oncolytic HSV-2 vectors.

Retroviruses present the classic approach for long-term gene therapy applications and the first human gene therapy trial involved implantation of autologous bone marrow cells transduced ex vivo with gamma retrovirus vectors [44]. More recently, attention has been paid to target dendritic cells (DCs) by engineering of vectors with DC-specific promoters or by retargeting vector tropism [45]. Also, transduction of hematopoietic stem cells has supported antigen-specific immune tolerance. In another immunotherapy approach, the low gene transduction efficiency of 50% of chimeric antigen receptor-expressing T (CAR-T) cells was improved to more than 90% by optimization of precultivation conditions and antibody stimulation [46]. The transduced CAR-T cells showed antigen-specific cytotoxic activity and secreted cytokines by antigen stimulation. Related to cancer therapy, the nonlytic amphotropid retroviral replicating vector (RRV) Toca 511 encoding yeast cytosine deaminase (CD) was delivered to tumors in orthotopic glioma models [47]. When combined with 5-fluorocytosine (5-FC), CD in infected tumor cells converts 5-FC to 5-fluorouracil (5-FU) leading to cell death. Intravenous or intracranial administration of Toca 511 provided long-term survival in immune-competent mice after combination treatment with 5-FC. Prolonged survival was also observed in animals with pre-existing immune response to the vector, which supports the potential of readministration. The self-inactivating gammaretroviral vector (SINfes.gp91s), containing the codon-optimized transgene (gp91(phax)) and the promoter for the X-linked form of the immunodeficiency named chronic granulomatous disease (CGD), was demonstrated to protect X-CGD mice from challenges with Aspergillus fumigatus [48].

In the case of lentivirus-based gene therapy, a lentiviral vector carrying the human pyruvate kinase deficiency (hPKD) promoter and the PKLR gene was employed for addressing the monogenic metabolic disease PKD caused by mutations in the pyruvate kinase isoenzymes L/R (PKLR) gene [49]. When mouse hematopoietic stem cells (HSCs) transduced with lentivirus were transplanted into myeoblated PKD mice, the erythroid compartment was normalized providing a corrected hematological phenotype and reversion of organ pathology. Furthermore, analysis of the genomic insertion sites for the lentivirus vector in transplanted hematopoietic cells indicated no presence of genotoxicity. Lentivirus vectors have also been subjected to gene therapy applications of RNA silencing in the CNS [50]. Related to Parkinsons disease (PD), the misregulation and overexpression of -synuclein leading to its accumulation in neurons was counteracted by lentivirus-based RNA interference (RNAi) in the human dopaminergic cell line SH-SY5Y and in neurons in rat striatum [51]. Moreover, in another approach, the PD-related transcriptional upregulation of the GABA-producing enzyme glutamate decaorboxylase 1 (GAD1) or GAD67 was successfully knocked down by lentivirus-mediated shRNA-miR expression in a rat model for PD, demonstrating normalized neuronal activity [52]. In the context of Alzheimers disease, lentivirus vectors have been applied for RNA silencing to knock down BACE1 attenuated amyloid precursor protein (APP) cleavage and -amyloid production, resulting in reduced neurodegeneration and behavioral deficits in an Alzheimers disease mouse model [53]. In another approach, lentivirus-based siRNA expression showed reduced tau phosphorylation and number of neurofibrillary tangles in an Alzheimers disease mouse model [54]. Furthermore, lentivirus vector-based delivery of shRNAs targeting the HIV-1 coreceptor CCR5 and the R-region of the HIV-1 long terminal repeat (LTR) has been evaluated in humanized bone marrow/thymus (hu-BLT) mice [55]. The outcome was efficient inhibition of HIV infection and might provide a potential therapy against HIV. In another approach, the Cal-1 anti-HIV lentiviral vector was evaluated in pigtailed macaques [56]. Cal-1 lentivirus demonstrated safe integration and preclinical safety.

Alphaviruses have been mainly applied in preclinical gene therapy studies for cancer treatment [57]. The particular feature is that alphavirus vectors can be delivered in the form of naked RNA, layered plasmid DNA vectors and recombinant replication-deficient or -proficient particles. In this context, local administration of a replication-proficient Semliki Forest virus (SFV) vector expressing EGFP, generated prolonged survival in mice with implanted A549 lung carcinoma xenografts [58]. In another study, SFV-IL-12-based therapy was evaluated in a syngeneic RG2 rat glioma model, which resulted in 87% reduction in tumor volume and significant extension of survival [59]. In attempts to target tumor cell replication, six micro-RNAs (miRNAs) were introduced into the SFV genome. Intraperitoneal administration of engineered SFV4-miRT124 particles in BALB/c mice resulted in glioma targeting, limited spread in the CNS and significantly prolonged survival rates [60]. Moreover, the naturally occurring oncolytic alphavirus M1 was demonstrated to selectively kill zinc-finger antiviral protein (ZAP)-deficient cancer cells and also showed high tumor tropism and potent oncolytic activity in a liver tumor model [61].

In the context of flaviviruses, the granulocyte macrophage colony-stimulating factor (GM-CSF) expressed from a Kunjin virus vector was subjected to intratumoral administration in mice with subcutaneous CT26 colon carcinoma [62]. The treatment resulted in a cure of more than 50% of injected mice; tumors were undetectable 18 days after Kunjin-GM-CSF administration. Likewise, treatment of B16-OVA melanoma tumors led to significant tumor regression after 5 days and the cure rate in mice reached 67% [62]. Moreover, subcutaneous injection of Kunjin-GM-CSF resulted in regression of CT26 lung metastasis in BALB/c mice.

Among rhabdoviruses, recombinant vesicular stomatitis virus (VSV) has been applied for preclinical gene therapy studies. The low seroprevalence in humans and robust heterologous expression profile have supported a number of vaccine approaches against human pathogens [63]. For instance, VSV vectors expressing HIV-1 Gag and Env elicited robust HIV-1 specific cellular and humoral immune responses in nonhuman primates [63]. Furthermore, vaccinated animals were protected against challenges with a pathogenic SIV/HIV recombinant. However, the neurovirulence of VSV vectors has remained an issue of concern leading to strategies of developing attenuated vectors [60]. In another approach, a chimeric VSV vector, where the VSV G envelope was replaced by a lymphocytic choriomeningitis virus glycoprotein (LCMV-GP), the chimeric vector presented no harm to normal brain cells, but efficiently eliminated brain tumor cells in several tumor models in vivo [64]. Moreover, safe systemic administration was confirmed in mice and no humoral activity against VSV was detected, which provided the basis for repeated systemic injections. In preparation for future clinical trials, the oncolytic VSV-IFN-NIS vector expressing interferon- (IFN) and sodium iodide transporter (NIS) was evaluated in preclinical rodent models [65]. For instance, dose-dependent tumor regression was demonstrated in C57BL1/KaLwRij mice implanted with syngeneic 5TGM1 plasmacytoma tumors. However, KAS6/1 xenografts regressed at all VSV doses tested in SCID mice. Moreover, purpose-bred dogs with naturally occurring tumors were subjected to a dose-escalation study with VSV-IFN-NIS [66]. The intravenous maximum tolerated dose (MTD) was determined to 1010 TCID50 with mild to moderate adverse events. The VSV genome disappeared rapidly and anti-VSV antibodies were detected 5 days after administration in the blood. However, no infectious virus was detected in the plasma, urine or buccal swabs. In another study, VSV-based expression of human mucin 1 (MUC1) provided significant reduction of tumor growth in mice with established pancreatic ductal adenocarcinoma xenografts [67]. Furthermore, combination of VSV-MUC1 and gemcitabine resulted in superior therapeutic efficacy.

Measles viruses have found a number of gene therapy applications, which have been evaluated in preclinical animal models. In this context, the oncolytic MV-Edm was engineered to express NIS, which is depleted in aggressive and radioiodine resistant anaplastic thyroid cancer (ATC) [68]. Treatment with MV-NIS confirmed NIS expression and enhanced tumor killing. In another approach, measles virus was engineered to express a yeast-based bifunctional suicide gene encoding cytosine deaminase and uracil phosphoribosyltransfrerase named super-cytosine deaminase (SCD) [69]. The chimeric protein is capable of converting the nontoxic prodrug 5-fluorocytosine (5-FC) into highly cytotoxic 5-fluorouracil (5-FU). Furthermore, 5-FU is directly converted into 5-fluorouridine monophosphate (5-FUMP), which addresses the issue of chemoresistance to 5-FU in cancer treatment. Transduction with MV-SCD showed replication and efficient lysis of human ovarian cancer cell lines and primary tumor cells. Moreover, precision-cut tumor slices from human ovarian cancer patients demonstrated efficient infection by MV-SCD. The MV-SCD also showed strong oncolytic activity in a mouse xenograft model of human hepatocellular carcinoma (HCC) [70]. Furthermore, MV-SCD generated long-term virus replication in tumor tissue and induced apoptosis-like cell death independent of intact apoptosis pathways. In another study, MV-SCD was administered intratumorally in combination with systemic 5-FU in a TFK-1 xenograft mouse model, which resulted in significant tumor reduction [71]. Moreover, tumor reduction and significant survival benefits were observed in a HuCCT1 xenograft model [71].

Newcastle disease virus (NDV) vectors have been frequently used in preclinical cancer therapy studies due to their oncolytic activity [72]. Although NDV vectors expressing IL-2 showed promise, comparative studies with the less toxic IL-15 have been conducted. Intratumoral injection of NDV-IL15 and NDV-IL2 in melanoma-bearing mice showed efficient suppression of tumor growth [72]. However, the 120 day survival rate was 12.5% higher after NDV-IL15 treatment than that of NDV-IL2. Likewise, the survival rate was 26.6% higher for NDV-IL15 treatment in a tumor rechallenge experiment. In another study, reverse genetics were employed on the oncolytic NDV D90 strain to generate recombinant NDVs carrying the GFP gene [73]. The rescued virus showed tumor-selective replication and induced apoptosis in tumor cells in athymic mice with implanted lung tumors. It has also been demonstrated that expression of IL-2 and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) enhanced inherent antineoplasticity by inducing apoptosis [74]. The NDV-TRAIL and the bifunctional NDV-IL2-TRAIL showed superior apoptotic function in comparison to NDV-IL2. Moreover, CD4+ and CD8+ proliferation was induced and expression of TFN- and IFN- antitumor cytokine expression was elicited. The NDV-IL2-TRAIL also exhibited prolonged survival in mice implanted with HCC and melanoma xenografts. In another study, the NDV Anhinga strain was applied for the expression of soluble TRAIL (NDV/Anh-TRAIL), which resulted in efficient suppression of HCC without significant cytotoxicity [75].

Coxsackieviruses have been used for several gene therapy applications [23]. For instance, the coxsackievirus B3 (CVB3) expressing the human fibroblast growth factor 2 (FGF2) was injected into ischemic hindlimbs of mice showing protection from ischemic necrosis [76]. The treatment improved the blood flow in ischemic limbs for more than 3 weeks. Moreover, the recombinant CVB3 showed a drastic decrease in virulence compared to wild type CVB3. Related to cancer, Coxsackievirus A21 (CAV21) expressing intercellular adhesion molecule-1 (ICAM-1) and decay-accelerating factor (DAF) reduced tumor burden in nonobese SCID mice implanted with melanoma xenografts [77]. A single administration of CAV21 was sufficient to provide efficient oncolysis and the systemic spread of CAV21 showed efficient regression in tumors distantly located from the site of viral injection. Furthermore, the same CAV21 vector was evaluated in SCID mice implanted with T47D and MDA-MB-231-luc breast tumor xenografts [78]. A single intravenous injection generated significant regression of pre-established tumors and also targeting and elimination of metastases. Furthermore, intravenous injection of CVA21 expressing ICAM-1 and DAF in combination with intraperitoneal injection of doxorubicin hydrochloride provided significantly enhanced tumor regression in comparison to either virus or drug alone in mice with implanted MDA-MB-231 tumors [79]. Related to prostate cancer, the low pathogenic enteroviruses, CVA21, CVA21-DAFv, and Echovirus 1 (EV1), were tested in SCID mice [80]. Systemic delivery induced regression of tumor xenografts and a therapeutic dose-response was obtained for escalating doses of EV1 in the LNCaP mouse model.

Finally, poxviruses have found several applications as gene therapy vectors. For instance, vaccinia virus vectors have demonstrated potential for treatment of pancreatic cancer [81]. In this context, the PANVAC system comprising of recombinant vaccinia and fowlpox viruses, carrying the tumor-associated antigens epithelial MUC-1 and carcinomebryonic antigen (CEA) as well as T cell stimulatory molecules, have been applied [82]. Sequential subcutaneous administration of the vectors has provided induced CEA and MUC-1 CTL responses in preclinical animal models. In the case of HCC, the light-emitting recombinant GLV-2b372 vaccinia virus was injected into HCC xenografts in the flank of athymic nude mice for assessment of tumor growth and inhibition of viral biodistribution [83]. It was demonstrated that flank tumor volumes decreased by 50% 25 days after injection, while tumor volumes increased by 400% in control mice. Related to prostate cancer, NIS expression from the GLV-1h153 vaccinia virus in combination with radiotherapy was evaluated in CD1 nude mice implanted with PC3 xenografts [84]. Combination therapy was superior to individual treatments both in xenograft and immunocompetent transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse models, demonstrating restricted tumor growth and improved survival rates. A vaccinia virus was engineered by mutating the F4L gene, the viral homologue of the cell-cycle-regulated small subunit of ribonucleotide reductase 2 (RRM2), which provided tumor-selective replication and cell killing [85]. It was confirmed that the F4L-mutated vector selectively replicated in immune-competent rat AY-27 and xenografted human RT122-luc orthotopic bladder cancer models, resulting in substantial tumor regression or complete ablation without causing any cytotoxicity. Moreover, antitumor immunity was established in rats cured of AY-27 tumors. Recently, a novel cowpox virus (CPXV) vector was engineered with a deletion of the thymidine kinase (TK) gene and insertion of the suicide gene FCU1, which is responsible for conversion of 5-FC into 5-FU and 5-FUMP [86]. Systemic administration of the modified CPXV vector showed accumulation in tumor cells and low infection and toxicity of normal cells. Moreover, intratumoral administration in U-87-MG glioblastoma and LoVo colon cancer models, induced relevant tumor growth inhibition.

A substantial number of clinical trials have been conducted or are currently in progress applying viral vectors (). For instance, the tumor-selective chimeric Enadenotucirev adenovirus vector was subjected to intravenous delivery in 17 patients with resectable colorectal cancer, non-small-cell lung cancer, urothelial cancer and renal cancer [87]. Tumor-specific delivery was observed in most tumor samples with no treatment-related serious adverse events.

Related to hemophilia, gene therapy has been employed already for three decades, mainly focusing on AAV-based vectors [88]. In addition to discovery of pre-existing neutralizing antibodies in animal models, clinical trials have revealed that liver transaminase levels are elevated and immune-related loss of transgene expression. The mechanism of the decrease in expression levels is not fully understood, but the use of different serotypes for consecutive administration of AAV has provided improved transgene expression [9], which has resulted in long-term expression of factors VIII (FVIII) and IX (FIX) and furthermore allows a cure of severe bleedings and joint damage associated with hemophilia. In this context, 11 hemophilia gene therapy clinical trials have been conducted and six ongoing phase I/II clinical trials have applied liver-directed AAV expressing either FVIII or FIX with some success [89]. Furthermore, stem cell-based lentiviral vector delivery has proven successful in establishing sustained high level FIX expression after differentiation of adipogenic, chondrogenic, and osteoblastic cells [90], which potentially can be applied for treatment of hemophilia B. Likewise, stem cell-based lentiviral gene therapy can provide life-long production of FVIII and the potential cure of hemophilia A [89].

The oncolytic HSV HF10 vector has been subjected to clinical trials in recurrent breast cancer, head and neck cancer, unresectable pancreatic cancer, refractory superficial cancer, and melanoma [42]. The studies demonstrated high safety and a low frequency of adverse effects in treated patients. Moreover, HF10 antigens were detected 300 days after immunization in pancreatic cancer patients. Combination therapy with HF10 and ipilimumab (anti-CTLA-4) showed a good safety profile and good antitumor efficacy in a phase II trial [42]. Related to retroviruses, a clinical trial in patients with recurrent high-grade glioma (HGG) is currently in progress with the Toca 511 retrovirus [47]. Moreover, Toca 511 was subjected to an open-label, ascending dose, multicenter phase I trial in patients with recurrent or progressive HGG [91]. The overall survival was 13.6 months and statistically better, relative to an external control group. Moreover, tumor samples from patients surviving more than a year demonstrated survival-related RNA expression in correlation with treatment-related survival. Currently, a phase II/III trial with Toca 511 is in progress [92]. In another approach, a gammaretroviral vector was employed for the treatment of chronic granulomatous disease (CGD), which relates to primary immunodeficiency, resulting in an impaired antimicrobial activity in phagocytic cells [48]. The phase I/II trial revealed that although bacterial and fungal infections were transiently resolved, clonal dominance and malignant transformations compromised the therapeutic effect, suggesting that alternative vectors should be considered for delivery [48]. In another cancer-related approach MV-NIS has been approved by the FDA for human clinical trials in myeloma patients, which could provide a potential strategy for targeting iodine-resistant ATC [66]. Oncolytic vaccinia viruses have also been subjected in a phase I clinical trial in 11 patients with refractory advanced colorectal or other solid cancers [93]. The study showed neither dose-related toxicity nor any treatment-related severe adverse events. However, a strong induction of inflammatory and Th1 cytokines indicated a potent mediation of potential immunity against cancer, which supports further trials with intravenously administered vaccinia virus in combination with expression of therapeutic genes, immune checkpoint blockade, or complement inhibitors. In another study applying poxviruses, the PANVAC-VF vaccine regimen composed of a priming dose of recombinant vaccinia virus and booster doses of recombinant fowlpox virus expressing CEA, MUC-1, and a triad of costimulatory molecules (TRICOM) was subjected to subcutaneous administration in patients with advanced pancreatic cancer [94]. The safety and ability of PANVAC-VF to induce antigen-specific T cells was demonstrated [80]. However, a phase III trial targeting patients with metastatic pancreatic cancer failed to meet the therapeutic targets and was terminated [95]. In another approach, a phase I trial for direct intratumoral injection of PANVAC-VF has generated some encouraging results [96].

In the context of HSV-based clinical trials, the oncolytic HSV M032 vector expressing IL-12 has been subjected to a phase I dose-escalating study in patients with recurrent or progressive malignant glioma [97]. Moreover, the HSV strain G207 lacking genes essential for replication in normal cells were evaluated in patients with recurrent glioblastoma multiforme [98]. After two doses of HSV G207 (totaling 1.15 109 pfu) no patients developed HSV encephalitis, but significant antitumor activity was observed. Furthermore, the study demonstrated safe multiple dose delivery including direct injections into the brain. In a phase I study in patients with recurrent/progressive HGG six of nine patients had stable disease of partial response and the median survival time was 7.5 months after a single-dose oncolytic HSV injection, indicating the potential for clinical response [99]. Furthermore, preclinical studies with HSV G207 have generated highly sensitive tumor killing, which support the initiation of the first-in-children study of intratumoral administration in children with recurrent or progressive supratentorial malignant tumors [100]. Alphaviruses have so far been subjected to only a limited amount of clinical trials. In this context, recombinant VEE replicon particles expressing the prostate specific membrane antigen (PSMA) were administered to patients with castration resistant metastatic prostate cancer in a phase I dose-escalation study [101]. The immunization showed no toxicity, but no PSMA-specific cellular immune response was detected with only weak signals detected by ELISA with a dose of 9 106 IU.

Similar results occurred when immunizations were performed with 3.6 107 IU. Despite the lack of clinical benefit and robust immune responses, immunizations elicited neutralizing antibodies, which encourages further dose optimization studies. In another approach, liposome-enveloped SFV vectors expressing IL-12 were subjected to systemic administration in a phase I study in melanoma and kidney carcinoma patients [102]. Intravenous injections provided a transient 5-fold increase of IL-12 in the plasma. Due to the encapsulation procedure, tumor targeting and protection against recognition by the host immune system was obtained, which also allowed repeated vector administration.

NDV has been used in a number of clinical trials [103]. For instance, NDV expressing multiple tumor-associated antigens (TAAs) has been demonstrated to provide long-term survival in phase II trials in patients with ovarian, stomach, and pancreatic cancer [104]. Furthermore, melanoma patients were immunized with NDV in a randomized double-blind phase II/III trial [105]. However, the study results suggested that there were no remarkable differences between the vaccinated individuals and those in the placebo group. In a phase II study 79 patients with solid tumors were subjected to intravenous administration of the NDV PV101 strain [106]. A lower dose of 12 109 pfu/m2 and an MTD of 12 1010 pfu/mL were applied, which resulted in objective response to the higher dose and progression-free survival ranging from 4 to 31 months. In another phase III trial, 335 patients with colorectal cancer were subjected to NDV immunotherapy [107]. It was demonstrated that vaccination with NDV provided prolonged survival and short-term improved quality of life.

Approaches on HIV gene therapy lentivirus vectors have been employed for targeting CCR5 by shRNA delivery [108]. The shRNAs were demonstrated to effectively inhibit CCR5 expression providing protection against HIV-1 infection in cell cultures [109]. Moreover, a self-activating lentiviral vector has been engineered to express a combination of the sh5 anti-HIV gene and the C46 antiviral fusion inhibitor peptide, which provided a synergistic effect on HIV-1 inhibition [108]. The promising results of preclinical studies triggered the first phase I clinical trial applying RNA interference to down-regulate CCR5 expression in HIV therapy [110].

Related to Coxsackieviruses, a phase I/II trial in melanoma patients with the CVA21 showed good tolerance, viral replication in tumors and increased antitumor activity [111]. The latter could be further enhanced by combination therapy with immune checkpoint blockade. In another phase II trial, CVA21 demonstrated induced immune cell infiltration in the tumor microenvironment of patients with melanoma [112]. Moreover, combination therapy of CVA21 and systemic pembrolizumab in a phase 1b study in melanoma patients showed a best overall response rate of 60% and stable disease in 27% of the patients [113]. Neither dose-limiting toxicity nor grade 3 or higher treatment-related adverse events were observed.

In the context of cystic fibrosis, a pseudotyped lentivirus vector with a fusion protein (F)/hemagglutinin-neuraminidase (HN) was optimized for promoter/enhancer sequences and evaluated in mice, and human airliquid interface (ALI) cultures in preparation for a first-in-man CF clinical trial [114]. The lentivirus vector carrying a hybrid cytosine guanine dinucleotide (CpG)-free CMV enhancer/elongation factor 1 alpha promoter (hCEF) expressed functional CFTR, retained 90100% transduction efficiency in clinically relevant delivery devices and showed acceptable toxicity and integration site profiles to support the initiation of a clinical trial in CF patients.

The first viral-based gene therapy drugs were approved some time ago in China [115]. In this context, oncolytic adenoviruses expressing the p53 gene (GendicineTM) [115] and AdH101 containing the E1b-55K deletion [116] are used for treatment of cancers with mutated p53 and head and neck cancer, respectively. GendicineTM has been used for 12 years in more than 30,000 patients with an exemplary safety record and has provided significantly better responses compared to standard therapies when combined with chemotherapy and radiotherapy [117]. Moreover, the progression-free survival times were significantly extended.

Furthermore, a second-generation oncolytic HSV vector expressing GM-CSF has been approved in the US and Europe for melanoma treatment [118,119]. Unfortunately, although the AAV-based Glybera drug was approved for treatment of the rare inherited disorder lipoprotein lipase deficiency, the high costs and limited demand forced the withdrawal from the market [120].

Additionally, several other viral-based drugs will most likely be on the market in the near future. For instance, oncolytic VV JX-594 (pexastimogene devacirepvec) for hepatocellular carcinoma treatment [121], Ad CG0070 expressing GM-CSF for bladder cancer [122], and the wild type retrovirus-based pelareorep (Reolysin) [123] for head and neck cancer are at late-stage development. Moreover, the third generation oncolytic HSV-1 G47, which was subjected to a phase II glioblastoma study [124], has been further designated as a Sakigake breakthrough therapy, which will provide priority reviewing and fast-track approval [118].

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Viral Vectors in Gene Therapy - PMC

Dive Brief:

Gene therapy holds great promise, with the potential to effectively cure an array of diseases. Already, the Food and Drug Administration has approved two of these medicines, Roche's Luxturna and Novartis' Zolgensma, the latter of which was developed at AveXis. Yet, as with most cutting-edge technologies, there have been challenges, among them that gene therapies can be costly to develop and are difficult to manufacture.

For young companies like Taysha, these challenges were eased by easy access to money. The last few years had seen the biotechnology sector flushed with record amounts of capital from venture firms and the public markets. Taysha, notably, priced shares at the top end of the company's estimated range when it went public in September 2020, raising $157 million in the process.

But investor sentiment toward biotechnology companies, which reached new heights in the early stages of the coronavirus pandemic, has worsened substantially in recent months. While the downtown has affected drugmakers in all areas of research, it's been hard on those developing gene therapies. In addition to Taysha, at least ten other gene therapy developers have announced layoffs, cost cuts or restructured programs since December.

Taysha's current priorities are to advance one program targeting Rett syndrome, which is in preclinical testing, and another focused on giant axonal neuropathy, which is currently in an early-stage study that should produce results later this year. The company noted, too, that it expects to hit milestones this year in programs for two types of Batten disease and a rare form of infantile epilepsy.

But elsewhere, Taysha is cutting back. A small trial testing one of its therapies against Tay-Sachs disease will stop enrollment, for example, though patients who were previously dosed will continue to be followed.

"To increase operational efficiency, activities for other ongoing clinical programs will be minimized and all additional research and development will be paused," Session said in a statement Thursday.

Taysha announced the layoffs and strategic changes alongside fourth quarter and full-year earnings. The company spent $132 million on research and development last year, and ultimately tallied a $173 million loss from operations. Session said that, with existing cash, debt financing and the newly implemented strategy, Taysha should have enough money to operate into the fourth quarter of 2023.

Taysha shares were up as much as about 3% Friday morning, before dipping down to near $6.50 apiece.

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Yet another gene therapy developer turns to layoffs - BioPharma Dive

Dive Brief:

Orchard recently secured an important agreement on reimbursement in the U.K. for Libmeldy, a gene therapy approved in Europe in December 2020 for children with early-onset metachromatic leukodystrophy. The company is working to expand newborn screening for the ultra-rare disease in other European countries, where two patients were recently treated under early access schemes.

The restructuring announced Wednesday puts Orchard's focus on Libmeldy, which the company hopes to submit for approval in the U.S. later this year or early next, as well as on two earlier gene therapies also for inherited neurometabolic diseases.

"In light of our experiences and knowledge gained in this current and rapidly evolving market environment for gene therapy, our plan is to concentrate resources on programs that have the potential to make a remarkable difference to patients while also providing sustainable value to the business to enable the achievement our long-term vision," said Bobby Gaspar, Orchard's CEO, in a statement.

While Orchard will keep active other research programs for future partnerships, the company will discontinue investment in gene therapies it was developing for rare primary immune deficiencies, including two currently in clinical testing. The path to an approval application in the U.S. for one of those gene therapies is now longer, Orchard said, citing feedback the company recently received from the Food and Drug Administration.

Orchard will also discontinue investment in Strimvelis, a gene therapy originally developed by GlaxoSmithKline that was approved in Europe six years ago. Since then, only 16 patients have received the therapy, which treats a rare immune condition known as ADA-SCID.

The cutbacks aren't the first time Orchard has laid off staff and discontinued research. The company announced layoffs soon after the COVID-19 pandemic began and, in June of last year, stopped developinganother treatment for ADA-SCID.

This time, Orchard has company. At least nine other cell and gene therapy developers have announced layoffs, cost cuts or altered their research plans since December. Bluebird bio, long a leading company in the field, warned investors earlier this month that there was "substantial doubt" about its ability to remain solvent over the next year.

With the expected cost savings, Orchard now anticipates being able to fund operations into 2024 and said it will seek "strategic alternatives" for its discontinued programs.

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Orchard turns to layoffs in cutting gene therapy research - BioPharma Dive

DUBLIN, April 04, 2022--(BUSINESS WIRE)--The "Nucleic Acid Based Gene Therapy Global Market Opportunities And Strategies To 2030, By Nucleic Acid Technology, Application, End User" report has been added to ResearchAndMarkets.com's offering.

The global nucleic acid-based gene therapy market grew from $1,391.9 million in 2015 to $4,726.8 million in 2020 at a compound annual growth rate (CAGR) of 27.7%. The market is expected to grow from $4,726.8 million in 2020 to $7,282.9 million in 2025 at a rate of 9.0%. The market is then expected to grow at a CAGR of 15.4% from 2025 and reach $14,909.6 million in 2030.

Growth in the historic period in the nucleic acid-based gene therapy market resulted from technological advances in synthetic biology, advances in combinatorial chemistry and bioinformatics, increased healthcare expenditure, rising pharmaceutical R&D expenditure, and rise in public-private partnerships.

The market was restrained by off-target specificity, challenges in nucleotide delivery to cells, instability of the nucleotides, inadequate reimbursements, challenges due to regulatory changes, low healthcare access, and limited number of treatment centers.

Going forward, a rise in healthcare expenditure, increasing prevalence of cancer and chronic diseases, rising geriatric population, rising geriatric population, increasing research and development spending and rising focus on gene therapy will drive the growth in the nucleic acid-based gene therapy market. Factors that could hinder the growth of the market in the future include high costs of therapy, stringent regulations, reimbursement challenges, and coronavirus pandemic.

The nucleic acid-based gene therapy market is segmented by technology into anti-sense and anti-gene oligonucleotides, SiRNA and RNA Interference, gene transfer therapy, ribozymes, aptamers, and others. The anti-sense and anti-gene oligonucleotides market was the largest segment of the nucleic acid-based gene therapy market segmented by technology, accounting for 92.90% of the total in 2020. Going forward, the others segment is expected to be the fastest growing segment in the nucleic acid-based gene therapy market segmented by technology, at a CAGR of 59.9% during 2020-2025.

Story continues

The nucleic acid-based gene therapy market is also segmented by application into oncology, muscular dystrophy/muscular disorders, rare diseases and others. The muscular dystrophy/muscular disorders market was the largest segment of the nucleic acid-based gene therapy market segmented by application, accounting for 61.4% of the total in 2020. Going forward, the oncology segment is expected to be the fastest growing segment in the nucleic-acid based gene therapy market segmented by application, at a CAGR of 18.1% during 2020-2025.

The nucleic acid-based gene therapy market is also segmented by end-user into hospitals and clinics, academic and research institutes. The hospitals and clinics market was the largest segment of the nucleic acid-based gene therapy market segmented by end-user, accounting for 85.0% of the total in 2020. Going forward, the academic and research institutes segment is expected to be the fastest growing segment in the nucleic-acid based gene therapy market segmented by end-user, at a CAGR of 9.8% during 2020-2025.

North America was the largest region in the global nucleic acid-based gene therapy market, accounting for 46.2% of the total in 2020. It was followed by the Western Europe, Asia Pacific and then the other regions. Going forward, the fastest-growing regions in the nucleic acid-based gene therapy market will be the Middle East and Eastern Europe where growth will be at CAGRs of 33.7% and 26.0% respectively. These will be followed by South America and Asia Pacific, where the markets are expected to register CAGRs of 21.0% and 20.4% respectively.

The global nucleic acid-based gene therapy market is fairly fragmented, with a large number of small players. The top ten competitors in the market made up to 16.40% of the total market in 2020. Major players in the market include Copernicus Therapeutics, Moderna Inc., Wave Life Sciences, Protagonist Therapeutics and Transgene.

The top opportunities in the nucleic acid-based gene therapy market segmented by technology will arise in the anti-sense and anti-gene oligonucleotides segment, which will gain $1,290.7 million of global annual sales by 2025. The top opportunities in the nucleic-acid based gene therapy market segmented by application will arise in the muscular dystrophy/muscular disorders segment, which will gain $1,000.2 million of global annual sales by 2025.

The top opportunities in the nucleic-acid based gene therapy market segmented by application will arise in the hospitals and clinics segment, which will gain $2,133.7 million of global annual sales by 2025. The nucleic acid-based gene therapy market size will gain the most in the USA at $915.0 million.

Key Topics Covered:

1. Nucleic Acid Based Gene Therapy Market Executive Summary

2. Table of Contents

3. List of Figures

4. List of Tables

5. Report Structure

6. Introduction

6.1. Segmentation By Geography

6.2. Segmentation By Technology

6.3. Segmentation By Application

6.4. Segmentation By End-User

7. Nucleic Acid Based Gene Therapy Market Characteristics

7.1. Market Definition

7.2. Segmentation By Nucleic Acid Technology

7.2.1. Anti-Sense and Anti-Gene Oligonucleotides

7.2.2. siRNA and RNA Interference

7.2.3. Gene Transfer Therapy

7.2.4. Ribozymes

7.2.5. Aptamers

7.2.6. Others

7.3. Segmentation By Application

7.3.1. Oncology

7.3.2. Muscular Dystrophy/ Muscular Disorders

7.3.3. Rare Diseases

7.3.4. Others

7.4. Segmentation By End-User

7.4.1. Hospitals And Clinics

7.4.2. Academic And Research Institutes

8. Nucleic Acid Based Gene Therapy Market Trends And Strategies

8.1. Global Research Initiatives And Funding

8.2. Integration Of Advanced Technologies In Gene Therapy

8.3. Increasing Partnerships And Acquisitions For Promoting Gene Therapy

8.4. Increasing Number Of Pipeline Studies And Drug Development

8.5. Growing Investments and Manufacturing Facility Expansion

8.6. Rising Focus On Gene Editing

9. Impact Of COVID-19 On The Nucleic Acid Based Gene Therapy Market

9.1. Introduction

9.2. Supply Chain Disruptions

9.3. Impact On Clinical Trials

9.4. Impact on Manufacturers and Activities

9.5. Conclusion

10. Global Nucleic Acid Based Gene Therapy Market Size And Growth

10.1. Market Size

10.2. Historic Market Growth, 2015 - 2020, Value ($ Million)

10.3. Forecast Market Growth, 2020 - 2025, 2030F, Value ($ Million)

11. Global Nucleic Acid Based Gene Therapy Market Segmentation

11.1. Global Nucleic Acid Based Gene Therapy Market, Segmentation By Technology, Historic And Forecast, 2015 - 2020, 2025F, 2030F, Value ($ Million)

11.2. Global Nucleic Acid Based Gene Therapy Market, Segmentation By Application, Historic And Forecast, 2015 - 2020, 2025F, 2030F, Value ($ Million)

11.3. Global Nucleic Acid Based Gene Therapy Market, Segmentation By End User, Historic And Forecast, 2015 - 2020, 2025F, 2030F, Value ($ Million)

Companies Mentioned

For more information about this report visit https://www.researchandmarkets.com/r/fisci7

View source version on businesswire.com: https://www.businesswire.com/news/home/20220404005624/en/

Contacts

ResearchAndMarkets.comLaura Wood, Senior Press Managerpress@researchandmarkets.com

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Nucleic Acid Based Gene Therapy Global Market Opportunities and Strategies to 2030 - ResearchAndMarkets.com - Yahoo Finance

WARREN, N.J.--(BUSINESS WIRE)--Tevogen Bio, a clinical stage biotechnology company specializing in the development of cell and gene therapies in oncology, neurology, and virology, today announced the strategic expansion of its leadership team with two new executives to support the biotechs rapid operational growth, manufacturing readiness, and the continued development and utilization of its next generation precision T cell platform technology.

New hires include:

-Stephen Chen, Chief Technical Officer

Stephen Chen, MBA, has more than 18 years of biotech industry experience. He was most recently Chief Operating Officer and Chief Technical Officer at NKGen Biotech where he led technical operations and the build out of the companys clinical GMP manufacturing facility. Previously, he was Senior Vice President of Quality and Technical Operations at NKGen Biotech. Before joining NKGen Biotech, he was Senior Director of Quality Assurance and Quality Control at ARMO Biosciences. Previously, he was Director of Quality Assurance at Kite Pharma. Chen started his career with over a decade of increasing responsibility in technical operations at Baxter BioScience. He earned an MBA from the University of Southern Californias Marshall School of Business and a Bachelor of Science in biochemistry/cell biology from the University of California, San Diego.

-Sadiq Khan, Global Commercial Lead

Sadiq Khan, MBA, brings over 30 years of commercial leadership, operations, and alliance management experience. Most recently, Sadiq served as Executive Director of Operations & Business Planning at BioCentriq where he played a key role in the creation of the CDMO specializing in cell and gene therapy manufacturing. Over the course of his career at Sanofi-Aventis and its predecessor companies, Sadiq successfully launched and managed multiple products in individual markets, regions, and globally. His experience covers over 12 therapeutic areas from specialty brands to blockbuster franchise with annual sales exceeding $1.4 billion. In addition to several country and regional commercial leadership roles in the Asia-Pacific region, he has held U.S. and global franchise leadership positions. Sadiq holds an undergraduate degree in mathematics and physics, and an MBA cum laude from University of Illinois at Chicago. He has been a regular guest speaker on topics related to biopharmaceutical commercialization, marketing, and alliance management at the Martin Tuchman School of Management at NJIT and the School of Engineering at Columbia University.

I have witnessed Tevogen Bios rapid growth and disruptive technological advances in the cell and gene therapy space and am excited to join a team who values scientific innovation and embraces a new brand of operational efficiency, Chen said.

Tevogens proprietary cell and gene therapy platforms with potential cures for hard-to-treat viral infections, neurological diseases, and cancers give me hope to finally offer accessible treatment options to the community. I am very excited to join this team of professionals where innovative ideas dont have to wait too long to become a reality, said Khan.

About Tevogens Next Generation Precision T Cell Platform

Tevogens next generation precision T cell platform is designed to provide increased specificity to eliminate malignant and viral infected cells, while allowing healthy cells to remain intact. Multiple targets are selected in advance with the goal of overcoming mutational capacity of cancer cells and viruses.

Tevogen believes its technology has the potential to overcome the primary barriers to the broad application of personalized T cell therapies: potency, purity, production-at-scale, and patient-pairing, without the limitations of current approaches. Tevogens goal is to open the vast and unprecedented potential of developing personalized immunotherapies for large patient populations impacted by common cancers and viral infections.

The companys lead product, TVGN-489, is currently in clinical trials for high-risk COVID-19 patients at Jefferson University Hospitals in Philadelphia. TVGN-489 is a highly purified, SARS-CoV-2-specific cytotoxic CD8+ T lymphocyte (CTL) product, which is designed to detect targets spread across the entire viral genome.

Tevogen recently announced it has completed dosing of the second cohort of patients in the proof of concept clinical trial of TVGN-489, marking the midway point of the trials planned four dosing levels. Trial details and recruitment information are available at Clinical Trials - Tevogen.

About Tevogen Bio

Tevogen Bio is driven by a team of distinguished scientists and highly experienced biopharmaceutical leaders who have successfully developed and commercialized multiple franchises. Tevogens leadership believes that accessible personalized immunotherapies are the next frontier of medicine, and that disruptive business models are required to sustain medical innovation in the post-pandemic world.

Forward Looking Statements

This press release contains certain forward-looking statements relating to Tevogen Bio Inc (the Company) and its business. These statements are based on managements current expectations and beliefs as of the date of this release and are subject to a number of factors which involve known and unknown risks, delays, uncertainties and other factors not under the Companys control that may cause actual results, performance or achievements to be materially different from the results, performance or other expectations implied by these forward-looking statements. Forward-looking statements can sometimes be identified by terminology such as may, will, should, intend, expect, believe, potential, possible, or their negatives or comparable terminology, as well as other words and expressions referencing future events, conditions, or circumstances. In any forward-looking statement in which the Company expresses an expectation or belief as to future results, there can be no assurance that the statement or expectation or belief will be achieved. Various factors may cause differences between the Companys expectations and actual results, including, among others: the Companys limited operating history; uncertainties inherent in the execution, cost and completion of preclinical studies and clinical trials; risks related to regulatory review and approval and commercial development; risks associated with intellectual property protection; and risks related to matters that could affect the Companys future financial results, including the commercial potential, sales, and pricing of the Companys products. Except as required by law, the Company undertakes no obligation to update the forward-looking statements or any of the information in this release, or provide additional information, and expressly disclaims any and all liability and makes no representations or warranties in connection herewith or with respect to any omissions herefrom.

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Tevogen Bio Expands Executive Leadership Team to Accelerate Operational Growth and Commercial Readiness - Business Wire

Venture capital firm 5AM Ventures, which has provided funding to a bevy of life sciences companies including Rallybio, Cleave Therapeutics, Cidara Therapeutics and Audentes Therapeutics, closed two new funds that will ultimately provide a combined $750 million in investments.

5AM Ventures, which has been making investments in the industry since 2002, closed Ventures VII, a $450 million early-stage fund, and Opportunities II, a $300 million fund aimed at late-stage companies. The $450 million aimed at early-stage companies will expand the firms goals of discovering and incubating startups and breakthrough life sciences companies. Both funds will be used to invest in existing 5AM Venture partners, as well as expand the firms portfolio of companies.

With the close of these two funds, Andrew Schwab, managing partner at 5AM Ventures, said the firm has raised more than $2.2 billion to invest in the life sciences industry. Schwab said 5AM is proud of the transformational impact that the companies it has invested in are making on the lives of patients as they attempt to develop new therapies and treatments for a multitude of disease indications.

One 5AM company, rare disease-focused Rallybio, has swiftly advanced its platform and financing capabilities, including an $80.6 million initial public offering last year. The company is enrolling patients in a Phase I study assessing RLYB212, a novel human monoclonal anti-HPA-1a antibody, being developed for the prevention of fetal and neonatal alloimmune thrombocytopenia (FNAIT). If RLYB212 proves successful in the clinic, it will become the first approved therapy for the prevention or treatment of FNAIT.

Audentes, an Astellas company now known as Astellas Gene Therapies, established a global Gene Therapy Center of Excellence last year. The company is exploring three gene therapy modalities: gene replacement, exon skipping gene therap, and vectorized RNA knockdown.

Last month, Cidara dosed the first patients in its Phase I study of CD388, an antiviral immunotherapy designed to deliver universal prevention of seasonal and pandemic influenza. The study is being conducted in collaboration with Janssen.

In addition to closing the two new funds, 5AM added two industry veterans to support its mission. Elliott Levy and Paula Soteropoulos joined the investment firm as a venture partner and strategic adviser, respectively. Levy previously served as head of global development at Amgen and before that, was president and head of specialty development at Bristol Myers Squibb. Levy serves on the boards of Omega Therapeutics and Nucana plc.

Soteropoulos was previously the chief executive officer of Akcea Therapeutics. She also held leadership roles at Moderna and Sanofi. At Moderna, she was head of cardiometabolic, rare diseases and strategic alliances, while at Sanofi Genzyme, she held numerous roles. Soteropoulos is a member of the boards of Rallybio, Ensoma, uniQure and Kyowa Kirin North America. She also serves on the advisory boards of Cheisi Rare Disease and Tufts Department of Chemical and Biochemical Engineering.

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5AM Ventures Pumps Another $750 Million into Life Sciences Industry - BioSpace

BioDrugs. 2017; 31(4): 317334.

1Janssen Research and Development, 200 McKean Road, Spring House, PA 19477 USA

1Janssen Research and Development, 200 McKean Road, Spring House, PA 19477 USA

1Janssen Research and Development, 200 McKean Road, Spring House, PA 19477 USA

2BiStro Biotech Consulting, LLC, Bridgewater, NJ 08807 USA

1Janssen Research and Development, 200 McKean Road, Spring House, PA 19477 USA

2BiStro Biotech Consulting, LLC, Bridgewater, NJ 08807 USA

Open AccessThis article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

There has been a resurgence in gene therapy efforts that is partly fueled by the identification and understanding of new gene delivery vectors. Adeno-associated virus (AAV) is a non-enveloped virus that can be engineered to deliver DNA to target cells, and has attracted a significant amount of attention in the field, especially in clinical-stage experimental therapeutic strategies. The ability to generate recombinant AAV particles lacking any viral genes and containing DNA sequences of interest for various therapeutic applications has thus far proven to be one of the safest strategies for gene therapies. This review will provide an overview of some important factors to consider in the use of AAV as a vector for gene therapy.

The discovery of DNA as the biomolecule of genetic inheritance and disease opened up the prospect of therapies in which mutant, damaged genes could be altered for the improvement of the human condition. The recent ability to rapidly and affordably perform human genetics on hundreds of thousands of people, and to sequence complete genomes, has resulted in an explosion of nucleic acid sequence information and has allowed us to identify the gene, or genes, that might be driving a particular disease state. If the mutant gene(s) could be fixed, or if the expression of overactive/underactive genes could be normalized, the disease could be treated at the molecular level, and, in best case scenarios, potentially be cured. This concept seems particularly true for the treatment of monogenic diseases, i.e. those diseases caused by mutations in a single gene. This seemingly simple premise has been the goal of gene therapy for over 40years.

Until relatively recently, that simple goal was very elusive as technologies to safely deliver nucleic acid cargo inside cells have lagged behind those used to identify disease-associated genes. One of the earliest approaches investigated was the use of viruses, naturally occurring biological agents that have evolved to do one thing, i.e. deliver their nucleic acid (DNA or RNA) into a host cell for replication. There are numerous viral agents that could be selected for this purpose, each with some unique attributes that would make them more or less suitable for the task, depending on the desired profile [1]. However, the undesired properties of some viral vectors, including their immunogenic profiles or their propensity to cause cancer have resulted in serious clinical adverse events and, until recently, limited their current use in the clinic to certain applications, for example, vaccines and oncolytic strategies [2]. More artificial delivery technologies, such as nanoparticles, i.e. chemical formulations meant to encapsulate the nucleic acid, protect it from degradation, and get through the cell membrane, have also achieved some levels of preclinical and clinical success. Not surprisingly, they also have encountered some unwanted safety signals that need to be better understood and controlled [3].

Adeno-associated virus (AAV) is one of the most actively investigated gene therapy vehicles. It was initially discovered as a contaminant of adenovirus preparations [4, 5], hence its name. Simply put, AAV is a protein shell surrounding and protecting a small, single-stranded DNA genome of approximately 4.8kilobases (kb). AAV belongs to the parvovirus family and is dependent on co-infection with other viruses, mainly adenoviruses, in order to replicate. Initially distinguished serologically, molecular cloning of AAV genes has identified hundreds of unique AAV strains in numerous species. Its single-stranded genome contains three genes, Rep (Replication), Cap (Capsid), and aap (Assembly). These three genes give rise to at least nine gene products through the use of three promoters, alternative translation start sites, and differential splicing. These coding sequences are flanked by inverted terminal repeats (ITRs) that are required for genome replication and packaging. The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40), which are required for viral genome replication and packaging, while Cap expression gives rise to the viral capsid proteins (VP; VP1/VP2/VP3), which form the outer capsid shell that protects the viral genome, as well as being actively involved in cell binding and internalization [6]. It is estimated that the viral coat is comprised of 60 proteins arranged into an icosahedral structure with the capsid proteins in a molar ratio of 1:1:10 (VP1:VP2:VP3) [6]. The aap gene encodes the assembly-activating protein (AAP) in an alternate reading frame overlapping the cap gene. This nuclear protein is thought to provide a scaffolding function for capsid assembly [7]. While AAP is essential for nucleolar localization of VP proteins and capsid assembly in AAV2, the subnuclear localization of AAP varies among 11 other serotypes recently examined, and is nonessential in AAV4, AAV5, and AAV11 [8].

Although there is much more to the biology of wild-type AAV, much of which is not fully understood, this is not the form that is used to generate gene therapeutics. Recombinant AAV (rAAV), which lacks viral DNA, is essentially a protein-based nanoparticle engineered to traverse the cell membrane, where it can ultimately traffic and deliver its DNA cargo into the nucleus of a cell. In the absence of Rep proteins, ITR-flanked transgenes encoded within rAAV can form circular concatemers that persist as episomes in the nucleus of transduced cells [9]. Because recombinant episomal DNA does not integrate into host genomes, it will eventually be diluted over time as the cell undergoes repeated rounds of replication. This will eventually result in the loss of the transgene and transgene expression, with the rate of transgene loss dependent on the turnover rate of the transduced cell. These characteristics make rAAV ideal for certain gene therapy applications. Following is an overview of the practical considerations for the use of rAAV as a gene therapy agent, based on our current understanding of viral biology and the state of the platform. The final section provides an overview for how rAAV has been incorporated into clinical-stage gene therapy candidates, as well as the lessons learned from those studies that can be applied to future therapeutic opportunities.

The main point of consideration in the rational design of an rAAV vector is the packaging size of the expression cassette that will be placed between the two ITRs. As a starting point, it is generally accepted that anything under 5kb (including the viral ITRs) is sufficient [10]. Attempts at generating rAAV vectors exceeding packaging cassettes in excess of 5kb results in a considerable reduction in viral production yields or transgene recombination (truncations) [11]. As a result, large coding sequences, such as full-length dystrophin, will not be effectively packaged in AAV vectors. Therefore, the use of dual, overlapping vector strategies (reviewed by Chamberlain et al.) [12], should be considered in these cases. An additional consideration relates to the biology of the single-stranded AAV-delivered transgenes. After delivery to the nucleus, the single-stranded transgene needs to be converted into a double-stranded transgene, which is considered a limiting step in the onset of transgene expression [13]. An alternative is to use self-complementary AAV, in which the single-stranded packaged genome complements itself to form a double-stranded genome in the nucleus, thereby bypassing that process [13, 14]. Although the onset of expression is more rapid, the packaging capacity of the vector will be reduced to approximately 3.3kb [13, 14].

AAV2 was one of the first AAV serotypes identified and characterized, including the sequence of its genome. As a result of the detailed understanding of AAV2 biology from this early work, most rAAV vectors generated today utilize the AAV2 ITRs in their vector designs. The sequences placed between the ITRs will typically include a mammalian promoter, gene of interest, and a terminator (Fig.). In many cases, strong, constitutively active promoters are desired for high-level expression of the gene of interest. Commonly used promoters of this type include the CMV (cytomegalovirus) promoter/enhancer, EF1a (elongation factor 1a), SV40 (simian virus 40), chicken -actin and CAG (CMV, chicken -actin, rabbit -globin) [15]. All of these promoters provide constitutively active, high-level gene expression in most cell types. Some of these promoters are subject to silencing in certain cell types, therefore this consideration needs to be evaluated for each application [16]. For example, the CMV promoter has been shown to be silenced in the central nervous system (CNS) [16]. It has been observed that the chicken -actin and CAG promoters are the strongest of these constitutive promoters in most cell types; however, the CAG promoter is significantly larger than the others (1.7kb vs. 800bp for CMV), a consideration to take into account when packaging larger gene inserts [15].

Schematic representation of the basic components of a gene insert packaged inside recombinant AAV gene transfer vector. AAV adeno-associated virus, ITR inverted terminal repeat

Although many therapeutic strategies involve systemic delivery, it is often desirable to have cell- or tissue-specific expression. Likewise, for local delivery strategies, undesired systemic leakage of the AAV particle can result in transduction and expression of the gene of interest in unwanted cells or tissues. The muscle creatine kinase and desmin promoters have been used to achieve high levels of expression, specifically in skeletal muscle, whereas the -myosin heavy chain promoter can significantly restrict expression to cardiac muscle [15, 17]. Likewise, the neuron-specific enolase promoter can attain high levels of neuron-specific expression [18, 19]. Often is the case, systemic delivery of AAV results in a significant accumulation in the liver. While this may be desirable for some applications, AAV can also efficiently transduce other cells and tissues types. Thus, in order to restrict expression to only the liver, a common approach is to use the 1-antitrypsin promoter [20, 21]. Finally, there are now technologies that have the ability to generate novel, tissue-specific promoters, based on DNA regulatory element libraries [22].

Over the course of the past 1015years, much work has been done to understand the correlation between codon usage and protein expression levels. Although bacterial expression systems seem to be most affected by codon choice, there are now many examples of the effects of codon engineering on mammalian expression [23]. Many groups have developed their own codon optimization strategies, and there are many free services that can similarly provide support for codon choice. Codon usage has also been shown to contribute to tissue-specific expression, and play a role in the innate immune response to foreign DNA [24, 25]. With regard to the gene of interest, codon engineering to support maximal, tissue-specific expression should be performed.

Additionally, terminator/polyadenylation signal choices, the inclusion of post-transcriptional regulator elements and messenger RNA (mRNA) stability elements, and the presence of microRNA (miRNA) target sequence in the gene cassette can all have effects on gene expression [26]. The human factor IX 3 UTR, for example, was shown to dramatically increase factor IX expression in vivo, especially in the context of additional cis regulatory elements [27]. Likewise, synthetic miRNA target sequences have been engineered into the 3 UTR of AAV-delivered genes to make them susceptible to miRNA-122-driven suppression in the liver [28]. Although there is much known about these individual components that needs to be considered when designing an AAV vector, the final design will most likely need to be determined empirically. It is not yet possible to know how a particular design will function by just combining the best elements together based on published reports, therefore considerable trial and error will eventually be required for deciding on the final construct. In addition, one also needs to consider the differences between in vitro and in vivo activity. Although it is possible to model rAAV expression in rodents, there is still significant concern about the translatability to humans.

AAV has evolved to enter cells through initial interactions with carbohydrates present on the surface of target cells, typically sialic acid, galactose and heparin sulfate [29, 30]. Subtle differences in sugar-binding preferences, encoded in capsid sequence differences, can influence cell-type transduction preferences of the various AAV variants [3133]. For example, AAV9 has a preference for primary cell binding through galactose as a result of unique amino acid differences in its capsid sequence [34]. It has been postulated that this preferential galactose binding could confer AAV9 with the unique ability to cross the bloodbrain barrier (BBB) and infect cells of the CNS, including primary neurons [35, 36].

In addition to the primary carbohydrate interactions, secondary receptors have been identified that also play a role in viral transduction and contribute to cell and tissue selectivity of viral variants. AAV2 uses the fibroblast/hepatocyte growth factor receptor and the integrins V5 and 51; AAV6 utilizes the epidermal growth factor receptor; and AAV5 utilizes the platelet-derived growth factor receptor. Recently, an uncharacterized type I membrane protein, AAVR (KIAA0319L), was identified as a critical receptor for AAV cell binding and internalization [37].

As a result of these subtle variations in primary and secondary receptor interactions for the various AAV variants, one can choose a variant that possesses a particular tropism and preferentially infects one cell or tissue type over others (Table). For example, AAV8 has been shown to effectively transduce and deliver genes to the liver of rodents and non-human primates, and is currently being explored in clinical trials to deliver genes for hemoglobinopathies and other diseases [38]. Likewise, AAV1 and AAV9 have been shown to be very effective at delivering genes to skeletal and cardiac muscle in various animal models [3946]. Engineered AAV1 is currently being explored as the gene transfer factor in clinical trials for heart failure, and has been approved for the treatment of lipoprotein lipase deficiency [47]. However, although different AAV vectors have been identified that preferentially transduce many different cell types, there are still cell types for which AAV has proven difficult to transduce.

Selected AAV vectors, known receptors, and known tropisms

With the strong desire to utilize AAV to deliver genes to very selective cell and tissue types, efforts to clone novel AAV variants from human and primate tissues have identified a number of unique capsid sequences that are now being studied for tropism specificities [48]. In addition, recombinant techniques involving capsid shuffling, directed evolution, and random peptide library insertions are being utilized to derive variants of known AAVs with unique attributes [4951]. In vivo-directed evolution has been successfully used to identify novel AAV variants that preferentially transduce the retinal cells of the eye, as well as other cell populations, including those in the CNS [50, 52, 53]. In addition, these techniques have been employed to identify novel AAV variants with reduced sensitivities to neutralizing antibodies (NAbs) [5457].

Alternatively, other investigators have inserted larger binding proteins into different regions of AAV capsid proteins to confer selectivity. For example, DARPins (designed ankyrin repeat proteins), portions of protein A, and cytokines, have all been engineered into the capsid of AAV for the purpose of greater cell specificity and targeting [58, 59]. Employing this concept, others have been able to selectively target AAV to tumors and CD4+ T cells, as examples of engineered tropism [60, 61].

As we continue to learn more about the biology of AAV with regard to the mechanisms involved in membrane translocation, endosomal escape, and nuclear entry, we will undoubtedly find opportunities to engineer unique properties into viral vectors through modulating one or more of these functions. For example, it has been hypothesized that surface-exposed serine and tyrosine residues could be phosphorylated upon viral cell entry, resulting in their ubiquitination and proteolytic degradation [6264]. Studies have shown that mutation of tyrosine to phenylalanine, which prevents this phosphorylation, results in dramatically improved transduction efficiencies [63]. Similar efforts have been made in attempts to limit the effects of NAbs, as discussed below.

The choice of a particular AAV to use as a gene transfer vector is heavily reliant on several critically important criteria: (1) which cell/tissue types are being targeted; (2) the safety profile associated with the delivered gene; (3) the choice of systemic versus local delivery; and (4) the use of tissue-specific or constitutively active promoters. As one gives careful consideration to these selection criteria, it is possible to narrow the choices of which AAVs (natural or engineered) to profile. Alternatively, one can begin the path of exploring fully engineered versions of AAV for truly selective cell targeting and optimized transduction. Because our understanding of AAV biology is in relative infancy, many of these efforts will remain empirical for quite some time as optimization for one activity could have a negative impact on another. Nonetheless, the future looks promising for this highly adaptable platform.

One of the appealing aspects of using rAAV as a gene transfer vector is that it is composed of biomolecules, i.e. proteins and nucleic acids. Fortunately, a full-package virus lacks engineered lipids or other chemical components that could contribute to unwanted toxicities or immunogenicities that may not be predictable or fully understood. In general, AAV has been shown to be less immunogenic than other viruses. Although not completely understood, one possible reason for this may hinge on the observation that certain AAVs do not efficiently transduce antigen-presenting cells (APCs) [65]. Additionally, unlike previous viral delivery strategies, rAAV does not contain any viral genes, therefore there will be no active viral gene expression to amplify the immune response [66]. Although AAV has been shown to be poorly immunogenic compared with other viruses (i.e. adenovirus), the capsid proteins, as well as the nucleic acid sequence delivered, can trigger the various components of our immune system. This is further complicated by the fact that most people have already been exposed to AAV and have already developed an immune response against the particular variants to which they had previously been exposed, resulting in a pre-existing adaptive response. This can include NAbs and T cells that could diminish the clinical efficacy of subsequent re-infections with AAV and/or the elimination of cells that have been transduced. It should be of no surprise that the formidable challenge is how to deliver a therapeutically efficacious dose of rAAV to a patient population that already contains a significant amount of circulating NAbs and immunological memory against the virus [67]. Whether administered locally or systemically, the virus will be seen as a foreign protein, hence the adaptive immune system will attempt to eliminate it.

The humoral response to AAV is driven by the uptake of the virus by professional APCs, and their presentation of AAV capsid peptides in the context of class II major histocompatibility proteins (MHCs) to B cells and CD4+ T cells [68, 69]. This leads to plasma cell and memory cell development that has the capacity to secrete antibodies to the AAV capsid. These antibodies can either be neutralizing, which has the potential to prevent subsequent AAV infection, or non-neutralizing. Non-NAbs are thought to opsonize the viral particles and facilitate their removal through the spleen [70].

Upon entry of the virus into target cells during the course of the natural infection process, the virus is internalized through clathrin-mediated uptake into endosomes [71]. After escape from the endosome, the virus is transported to the nucleus where the ITR-flanked transgene is uncoated from the capsid [72]. The pathway and mechanism of AAV intracellular transport and processing is not fully understood, and there are quite a few areas of debate with regard to current understanding. The most current hypothesis is that following endosomal escape, capsid breakdown and uncoating occurs after subsequent nuclear translocation. However, it is thought that cytosolic ubiquitination of the intact virus can occur during transport to the nucleus [73]. This would be a critical step in directing capsid proteins to the proteasome for proteolytic processing into peptides for class I MHC presentation. This hypothesis is supported by data in which proteasome inhibitors, or mutations in capsid residues that are sites for ubiquitination, can limit class I presentation and T-cell activation [7376]. However, apparent differences have been observed for T-cell activation to different AAV variants with significant sequence identity. At this time, it is unclear whether this is due to subtle capsid sequence differences and susceptibility to MHC I presentation or differential cellular processing that is innate to the different AAV variants, or simply due to contaminants in vector preparations [76].

In addition to an adaptive immunological reaction to the capsid of AAV, the transgene can elicit both an adaptive and an innate response. If the transgene encodes a protein that can be recognized as foreign, it too can generate a similar B- and T-cell response. For example, in replacement therapy applications in which the protein to be replaced is the consequence of a null genotype, the immune system will have never selected against precursor B and T cells to that protein [70, 77]. Likewise, if the transgene is an engineered variant, the engineered sequence can be recognized as foreign. Even the variable regions of antibodies can activate an adaptive response that can result in deletion of target cells that are expressing transgene as a result of AAV delivery. Finally, a transgene with a significant number of CpG dinucleotides can activate innate responses through toll-like receptor (TLR) molecular pattern receptors [78].

Pre-existing immunity to AAV, especially the presence of circulating NAb, can have a dramatic effect on AAV clinical efficacy. To date, this represents one of the biggest therapeutic challenges to the use of systemically delivered AAV, and is thought to be one of the factors in early clinical failures [79]. Pre-existing immunity to AAV can often be overcome by selecting a particular AAV variant that has not circulated throughout the human population, and, therefore, does not have any memory responses elicited against it, including NAbs and T cells [80]. Additionally, some of the AAV evolution technologies discussed above have been used to identify AAVs that are resistant to the effects of NAbs [50, 57]. Although not optimal, it is possible to prescreen subjects for the presence of NAbs to the particular AAV variant to be used. In addition, the impact of this immunological response can sometimes be minimized by the particular route of administration employed for the particular therapeutic strategy, as discussed in Sect. 6 [80].

Like most biotherapeutics, AAV needs to be produced in a living system (Fig.). The parallels with recombinant antibody production during the 1990s and 2000s, with regard to the upstream challenges of robust production levels, are important to understand where the industry currently is, and where we need to strive to be.

Overview of AAV production/purification. Cell platform: HEK-293T, Sf9, or other suitable cell system can be grown on a small scale on 150mm tissue culture-treated culture dish, hyperflasks, or shake flasks. Cells are then transfected with adenovirus helper virus, rep/cap, and ITR-transgene plasmids for 293T, or infected with baculovirus for Sf9. Producer lines with integrated expression of rep/cap and ITR-transgene can be infected with adenovirus and grown to scale. Scale-up: For larger-scale culture volumes, virus can be produced in roller bottles, continuous perfusion, or WAVE Bioreactor systems. Purification/polishing: Affinity or heparin chromatography are optimal for isolation of virus from culture supernatants with or without cell pellet harvesting. Benzonase/DNAse treatment of eluted virus is required for removal of extraviral DNA contamination, followed by anion-exchange chromatography to fractionate empty vs. full AAV particles. QC/release: Upper left of far right panel: image depicts a silver stain analysis of culture FT next to affinity/anion exchange purified AAV (pure). The three bands represent the viral capsid proteins VP1, VP2, and VP3. Upper right of far right panel: Dynamic light scattering analysis of purified AAV1 indicates a uniform particle distribution of approximately 2530nM. Bottom half of far right panel: Analytical ultracentrifugation can resolve the proportion of empty vs, full particles of purified material. Additional assays that should be employed are digital drop polymerase chain reaction for determining titer in GC/mL, cryo or transmission electron microscopy for visual representation of purified particles, endotoxin testing, and other assays to evaluate the presence of residual host-cell protein contamination. AAV adeno-associated virus, FT flow-through, GC genome copies, rep/cap replication/capsid, QC quality control

Current methods to produce rAAV are still expensive despite years of research (Table). The most widely used platform for producing rAAV involves transfecting HEK293 cells with either two or three plasmids; one encoding the gene of interest, one carrying the AAV rep/cap genes, and another containing helper genes provided by either adeno or herpes viruses [6]. While most robust production rates have been achieved with adherent cells in either roller bottles or cell stacks, similar rates are now achievable in suspension-adapted HEK293 cells (Table). Production rates of approximately 105 genome copies (GC)/cell are now common, resulting in 1014 GC/L [81]. While this has proven to be sufficient to support early clinical trials, and could supply marketed product for small patient population indications, the deficiencies in scalability with this platform are a significant limitation [82, 83]. As one could surmise, successfully delivering three plasmids to one cell is a relatively inefficient process. For larger-scale manufacturing efforts, transient delivery of plasmid requires excess quantities of DNA, adding to the overall cost of production and purification. Moreover, transient delivery of rep/cap genes in the presence of helper genes can also contribute to product heterogeneity, including AAV vectors lacking a transgene. These empty capsids represent a significant proportion of virus produced in transient transfection assays. Thus, it is critically important to develop robust analytical quality control (QC) methods that are able to distinguish between these viral variants in order to ensure similarities between production lots [82, 83].

Current manufacturing platforms being employed to generate rAAV for clinical use

In three other AAV manufacturing platforms, one or more genetic components for the AAV manufacturing has been integrated into the genome of mammalian or insect production cell lines. While most viral helper genes needed for AAV production cannot be stably transfected, the adenoviral E1a and E1b genes are exceptions. These genes have been used to transform HEK293 cells, however they induce expression of the AAV rep gene, which is toxic to mammalian and insect cells [84, 85]. Hence, two different approaches have been used to develop mammalian cell lines. The first uses co-infection of BHK cells with two replication-defective HSVs engineered to encode the ITR-flanked transgene and the rep/cap genes. The second is based on stable producer cell lines in HeLa cells carrying the ITR-flanked transgene and the rep/cap genes. Rep proteins are not expressed in these cells since HeLa carries no adenoviral genes. However, infection with wild-type adenovirus is required for AAV production. The inclusion of replication-competent viral agents into a production process is a concern that needs to be addressed and also requires additional steps during the downstream processing [82, 83].

More recently, the Sf9 insect cell system in combination with baculovirus infection has been utilized to produce bulk quantities of rAAV. In this system, two or three baculovirus particles may be used to infect the Sf9 cells and initiate AAV production. In one example, one virus contains the rep gene, a second contains the cap gene, and the final virus carries the ITR-flanked gene of interest. In an alternative system, the Sf9 cells can be engineered to have the ITR-flanked gene of interest integrated into their genome, upon which production is initiated with only two baculovirus preps [81, 82]. A further improvement has recently been shown whereby the rep/cap genes are stably integrated into the Sf9 cell line genome, but are under the control of a promoter/enhancer that is induced by subsequent baculovirus infection. In this system, infection can occur, with only one baculovirus containing the ITR-flanked gene of interest, simplifying the system significantly [86, 87].

Production levels of approximately 105 GC/cell and 1015 GC/L have routinely been achieved with these Sf9 systems. Because of their ease of manipulation and their ability to grow to very high cell densities, the Sf9 system is rapidly becoming the platform of choice for AAV manufacturing. Concerns regarding baculovirus instability and differences in post-translational modifications between mammalian and insect cell systems are now beginning to be understood and controlled. These concerns are offset by the fact that baculovirus cannot efficiently infect mammalian cells which makes it inherently safer then other viral-based production systems [8183, 86, 87].

Unlike antibody manufacturing that relied on a single protein A-based purification platform early in the development of the downstream process, AAV is still rapidly evolving in that area. The products of an AAV production run will contain not only cellular debris (protein/lipids/nucleic acids) but also two main populations of AAV particles: particles that contain (full capsids) or those lacking (empty capsids) the ITR-flanked transgene. Although still widely debated in the field, the presence of empty capsids represents another contaminant that must be removed or controlled. Initial attempts to separate these two populations originally relied on the cumbersome and non-scalable method of density ultracentrifugation. In addition to the scalability issue, there are also concerns about the physiochemical effects of this method on the particles. Regardless, this method is still employed by many organizations as either a primary or secondary step in AAV purification [83].

Current technologies utilizing various affinity resins and/or ion exchange chromatography are being adopted by the industry. As mentioned above, AAV uses cell membrane-associated carbohydrates as the primary cell receptor for transduction. This affinity for carbohydrates can be exploited as an initial capture step in AAV purification. Indeed, heparin columns are frequently used in many downstream processing steps for AAV [88]. However, because of the lack of specificity, alternative affinity columns based on AAV-specific binding proteins such as scFvs and antibody single domains from llamas (camelids) have started to dominate the field. Improvements in generating these AAV-specific resins confers many advantages in downstream purification. These resins have the ability to bind to more than one AAV variant, have very high binding capacities (>1014GC/mL resin), and are stable against harsh clean-in-place and regeneration methods, making them suitable for use multiple times. Some of these commercial resins are already Good Manufacturing Practice (GMP) compliant, making them ideal for downstream manufacturing at commercial scales. Polishing steps using anion exchange chromatography are now routinely included after affinity capture steps, and can efficiently separate full capsids from empty capsids [8992].

As with any new therapeutic platform, and, again, similar to antibody-based therapeutic evolution, details on product specification and regulatory requirements are still evolving. With still very limited clinical experience, the impact of empty particles, host-cell impurities, post-translational modifications from different production platforms, fidelity of the packaged transgene, capsid ratio integrity, and probably many other specifications are still not known. However, over time, and as more clinical experience is gained, the field will be able to better relate these details to product performance and safety [83].

The use of rAAV as a delivery vector for gene therapies has been rapidly gaining interest over the past 35years. As approvals begin to increase (see Sect.6), efforts to optimize and maximize clinical manufacturing technologies will see a burst of activity. This will most likely mirror what occurred with antibody therapeutics in the 1990s and 2000s, in which early technologies were quickly overcome by next-generation technologies, resulting in significant cost savings and increased clinical supplies.

AAV has been shown to be a very stable vector able to withstand wide temperature and pH changes with little to no loss in activity [93]. To date, the only limitation seems to be the concentration with which it can be formulated, currently maximized around 51013 particles per milliliter [83]. With the resurgence in clinical use, this formulation limit will most likely be overcome in the near future. However, the robust stability of these vectors provides ample opportunities to attempt different routes of administration and specialized delivery strategies (Table).

Selected examples of more than 50 clinical candidates employing rAAV

Other than the European Medicines Agency (EMA)-approved AAV-based product alipogene tiparvovec (Glybera), the most advanced current clinical trial using AAV is sponsored by Spark Therapeutics and utilizes local injection of AAV2 into the eye for inherited retinal diseases (voretigene neparvovec-RPE65) (Table) [94]. Phase III studies have just been completed on this candidate and a Biologics License Application (BLA) submission is expected this year. This type of local delivery has proven to be safe and efficacious, but requires specialized surgical techniques and/or devices to deliver the vector [94, 95]. Similar strategies are being conducted by Applied Genetic Technologies Corporation (AGTC), targeting X-linked retinoschisis and achromatopsia, X-linked retinitis pigmentosa, and age-related macular degeneration. These programs are at various stages of development, with the most advanced for X-linked retinoschisis and achromatopsia in phase I safety studies (http://www.AGTC.com) (Table).

Several clinical trials are being run in which systemic administration is being used to target the liver, a tissue that is readily accessible through this route of administration and a tissue type that is readily transduced by many well-understood AAV variants [96]. These trials are mostly for monogenic, inherited diseases, in which the goal is gene replacement for defective genes, including those mutated in hemophilia A and B. Currently, these trials are in phase I/II, and are sponsored by academic groups, as well as biopharmaceutical companies such as Spark Therapeutics (SPK-9001, SPK-8011), Sangamo Therapeutics (SB-525), UniQure (AMT-060), Dimension Therapeutics (DTX101, DTX201), and Biomarin (BMN 270) (Table) [97]. Unlike local administration to the eye, which is considered an immune-privileged site that might not be affected by the existence of NAbs, systemic administration will require patient stratification for patient NAb levels. In addition, the possibility for re-administration becomes very difficult, should the need arise [80]. Although rare, there have been reports of rAAV vector integration into animal model genomes with subsequent genotoxicities [98, 99]. In addition, AAV genome sequences have been found in human hepatocellular carcinoma samples near known cancer driver genes, although at a low frequency [100]. There is an ongoing debate on these findings regarding cause and effect, and mouse/human translation. Regardless, hepatocellular, as well as other tissue genotoxicity, will need to be monitored in the course of AAV clinical development.

Another common delivery strategy is direct intramuscular injections. The only approved AAV gene therapy in Europe (Glybera) is an AAV1 encoding the gene for lipoprotein lipase deficiency [47, 101]. Skeletal muscle has been shown to be a target tissue type that is efficiently transduced by many AAV variants [39]. Once transduced, the muscle cells serve as a production site for protein products that can act locally or systemically, as is the case with Glybera. As a result of the low cellular turnover rate of the muscle cells, the transduced AAV gene product will be maintained in these cells as an episome for years, as has been shown in many studies in non-human primates [39]. Consequently, a single-dose regimen of an intramuscularly-delivered product may never need to be readministered unless there is significant damage or immune clearance of the transduced cells. This strategy is also being employed by Adverum and AGTC for 1-antitrypsin deficiency, as well as for certain muscular dystrophies (Table) [97].

Direct CNS administration is being utilized for Parkinsons disease, as well as various inherited diseases such as Batten disease, Canavan disease, and mucopolysaccharidosis (MPS) IIA and IIB, as well as MPS IIIa and MPS IIIb (Sanfilippo syndromes type A and type B, respectively). Phase I/II studies for these diseases using a variety of AAV variants, including AAV2, AAVrh10, and AAV9, are currently ongoing by various academic groups and biopharmaceutical companies, such as Abeona Therapeutics (ABO-101, ABO-102, ABO-201, ABO-202) [97, 102, 103]. Delivery strategies range from direct intraparenchymal administration into particular areas of the brain, intracerebroventricular, and cisternal and lumbar intrathecal routes [102]. The decision on the best route of administration is intimately related to the disease and affected areas. For example, for Parkinsons disease, according to our current understanding of disease pathogenesis and therapeutic strategies, direct injection into the putamen, substantia nigra or striatum is thought to be required. Similarly, for diseases that affect larger areas of the brain, such as Canavan disease or MPS, direct injection into the cerebellum is thought to be most beneficial [102, 103].

Alternatively, administration directly into the cerebrospinal fluid through an intrathecal route can result in wide CNS biodistribution, which is thought to be necessary for diseases such as spinal muscular atrophy (SMA) and Alzheimers disease [102106]. An alternative to cerebral spinal fluid (CSF)-based routes is the use of systemic administration of AAV variants that have been shown to cross the BBB. AAV9 has been shown to transcytosis across the BBB and transduce large sections of the CNS [36, 104, 107, 108]. This approach is currently being explored in the clinic for the treatment of SMA by AveXis (AVXS-101).

Neurodegenerative diseases represent a particular devastating health problem for which there is significant unmet medical need. These diseases of the CNS have proven to be very difficult to treat as a result of our poor understanding of their etiology and difficulty getting efficacious agents across the BBB. With regard to Alzheimers disease, although there is still some disagreement in the field, idiopathic amyloid plaque formation or generation of neurofibrillary tau tangles (NFTs), both of which are thought to be neurotoxic, are still the prevailing hypotheses behind the mechanism of many of these neuropathologies. Attempts to clear these plaques with plaque-specific antibodies have shown signs of limiting this process in animals and early-stage clinical trials [109, 110]; However, larger studies have all shown to be inconclusive at best, or failures at worst. It is unclear if these failures were because the plaque hypothesis is wrong, or if there was inefficient CNS exposure to the antibody therapeutic [110, 111]. Alternative strategies taking advantage of the safety and persistence of AAV would utilize either local administration of antibody-encoding AAVs directly to the CNS, or systemic delivery of AAVs that can cross the BBB, resulting in significantly higher CNS exposure levels of the antibody [112].

Local delivery of AAV to cardiac muscle for heart failure has been attempted in various clinical trials. In one case, Celladon failed in their attempt to deliver SERCA2A directly to the heart, and, in a second case, there is an ongoing program sponsored by UniQure to deliver S100A directly to the heart that is currently still in preclinical development [46, 113115]. Although it is not thoroughly clear why Celladon failed in the clinic, and why one would expect UniQure/BMS to succeed, there are significant differences in the delivery methods used by the two programs and the target gene delivered. Celladon used intracoronary infusion to deliver their AAV1 SERCA2A gene product, whereas UniQure is using retroinfusion and left anterior descending (LAD) coronary occlusion [41, 115]. This procedure is thought to better localize and restrict the delivered AAV9 S100A gene product to better target the heart tissue of interest. The reality of this suspected benefit will be realized in the clinic in the coming years.

Aerosolized AAV for inhaled pulmonary delivery was utilized in some of the earliest trials for cystic fibrosis (CF). Although none of these trials resulted in significant benefit or showed much of a pharmacodynamic response, they did help to show the safety of AAV when administered via this route [116118]. More importantly, the pathophysiology of CF, molecular biology of the CF transmembrane conductance regulator (CFTR) gene, and the target cell population for this type of indication exposed some key considerations when using AAV [117]. Congestion of the airways in these patients can limit AAV biodistribution after delivery, thus attenuating robust transduction [118]. In addition, the CFTR gene is over 4kb in size, putting it at the upper limit of the packaging capacity of AAV after also considering a required promoter and terminator. Finally, CFTR is expressed by the submucosal glands, which may be difficult to target efficiently [116, 117]. Nonetheless, these early efforts proved that AAV can safely deliver genes to the lung, which might be an ideal strategy for other diseases, such as influenza and other infectious diseases of the lung [119].

The field is just beginning to explore localized delivery of AAV for gene therapy applications. The stability of the virus and broad tropism for many different cell and tissue types make them ideal for most applications. There appears to be at least one AAV variant option for every tissue type of interest, with engineering and novel AAV discovery efforts sure to identify and create AAV variants with very specialized functions on demand. These efforts will undoubtedly result in new therapeutic strategies for many new indications.

The transfer of genes and other nucleic acids into cells has been a research tool in the laboratory for more than four decades. However, it was our growing understanding of the genetic components underlying certain diseases that has driven the search for true gene therapies. Progressively, research in other areas have identified other potential opportunities in which gene delivery could be applied therapeutically. In addition, limitations with current small molecule and protein therapeutic platforms have also driven the search for alternative therapeutic platforms that accommodate those limitations [120, 121]. Gene therapies accommodate all of those limitations, especially around target accessibility. As a result, the search for safe and effective gene delivery technologies has been a major focus in pharmaceutical research and development, and will hopefully represent a paradigm shift in how we approach disease-state intervention.

AAV was discovered over 50years ago and has since become one of the leading gene delivery vectors in clinical development. As a result of its unique biology, simple structure, and no known disease associations, AAV could become the vector of choice for most gene therapy applications. Gene therapy using rAAV has been demonstrated to be safe and well-tolerated in virtually every clinical setting in which it has been used. These studies, along with basic research on its biology, have revealed many facets of this vector that can be applied to future efforts.

Among the critical parameters to be considered are vector design, capsid selection, desired target cell and tissue type, and route of administration. The transgene to be delivered optimized for expression, the right AAV variant with an appropriate capsid for target cell transduction and immunoreactivity profile, and the appropriate delivery approach to maximize target tissue exposure while limiting off-tissue exposure are key focal points for AAV-based therapies.

All of these variables will be dictated by the overall therapeutic strategy which will be influenced by our understanding of the pathobiology of the disease to be treated. Will the transgene have the desired effect? Is the target cell driving the disease state? Is the turnover rate of the target cell high, requiring repeat dosing? This cannot be emphasized enough; without a strong understanding of the mechanisms driving the disease state, it will not be possible to design, discover, and develop the right gene therapeutic. Better designed trials, optimized vector construction, and novel AAV variants will certainly result in future regulatory approvals and improvements on patient outcomes and health.

Michael F. Naso, Brian Tomkowicz, and William L. Perry III are employees of Janssen Research and Development. William R. Strohl has no conflicts of interest to declare.

No funding was received for the preparation of this review.

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Adeno-Associated Virus (AAV) as a Vector for Gene Therapy ...

Researchers in the School of Veterinary Medicine and colleagues have developed a gene therapy that restores dim-light vision in dogs with a congenital form of night blindness, offering hope for treating a similar condition in people.

People with congenital stationary night blindness (CSNB) are unable to distinguish objects in dim-light conditions. This impairment presents challenges, especially where artificial lighting is unavailable or when driving at night.

In 2015, researchers from Penns School of Veterinary Medicine learned thatdogs could developa form of inherited night blindness with strong similarities to the condition in people. In 2019, the teamidentified the gene responsible.

Today, in the journalProceedings of the National Academy of Sciences,theyve reported a major advance: a gene therapy that returns night vision to dogs born with CSNB. The success of this approach, which targets a group of cells deep in the retina called ON bipolar cells, charts a significant step toward a goal of developing a treatment for both dogs and people with this condition, as well as other vision problems that involve ON bipolar cell function.

Dogs with CSNB that received a single injection of the gene therapy began to express the healthy LRIT3 protein in their retinas and were able to ably navigate a maze in dim light. The treatment also appears lasting, with a sustained therapeutic effect lasting a year or longer.

The results of this pilot study are very promising, saysKeiko Miyadera, lead author on the study and an assistant professor at Penn Vet. In people and dogs with congenital stationary night blindness, the severity of disease is consistent and unchanged throughout their lives. And we were able to treat these dogs as adults, between 1 and 3 years of age. That makes these findings promising and relevant to the human patient population, as we could theoretically intervene even in adulthood and see an improvement in night vision.

In the earlier work, the Penn Vet team, working in collaboration with groups from Japan, Germany, and the United States, discovered a population of dogs with CSNB and determined that mutations in theLRIT3gene were responsible for the dogs night vision impairment. The same gene has been implicated in certain cases of human CSNB as well.

This mutation affects the ON bipolar cells function, but, unlike in some blinding diseases, the overall structure of the retina as a whole remained intact. That gave the research team hope that supplying a normal copy of theLRIT3gene could restore night vision to affected dogs.

Yet while Penn Vet researchers from theDivision of Experimental Retinal Therapieshave developed effective gene therapies for a variety of other blinding disorders, none of these earlier treatments has targeted the ON bipolar cells, located deep within the middle layer of the retina.

Weve stepped into the no-mans land of the retina with this gene therapy, saysWilliam A. Beltran, a coauthor and professor at Penn Vet. This opens the door to treating other diseases that impact the ON bipolar cells.

The researchers overcame the hurdle of targeting these relatively inaccessible cells with two key findings. First, through a rigorous screening process conducted in collaboration with colleagues at the University of California, Berkeley, led by John Flannery and at the University of Pittsburgh led by Leah Byrne, they identified a vector for the healthyLRIT3gene that would enable the treatment to reach the intended cells. And, second, they paired the healthy gene with a promoterthe genetic sequence that helps initiate the reading of the therapeutic genethat would act in a cell-specific fashion.

Prior therapies weve worked on have targeted photoreceptors or retinal pigment epithelium cells, says coauthorGustavo D. Aguirre, a Penn Vet professor. But the promoter we use here is very specific in targeting the ON bipolar cells, which helps avoid potential off-target effects and toxicity.

The researchers suspect that restoring the functionalLRIT3gene enables signals to cross from the photoreceptor cells to the ON bipolar cells. LRIT3is expressed at the finger tips of these cells, says Beltran. Introducing this transgene is essentially allowing the two cells to shake hands and communicate again.

An open question is whether targeting both photoreceptor cells and ON bipolar cells together could lead to even greater improvements in night vision. Other research groups studying these conditions in mice have targeted the therapy to photoreceptor cells and found some vision to be restored, suggesting a possible path to enhance the effects of gene therapy.

And while the therapy enabled functional recoverydogs were able to navigate a maze when their treated eye was uncovered but not when it was coveredthe healthy copy of the gene was only expressed as much as 30% of ON bipolar cells. In follow-up work, the researchers hope to augment this uptake.

"We had great success in this study, but we saw some dogs get better recovery than others, says Miyadera. Wed like to continue working to maximize the therapeutic benefit while still ensuring safety. And weve seen that this treatment is durable, but is it lifelong after one injection? Thats something wed like to find out.

The team also plans to amend the therapy to use the human version of theLRIT3gene, a necessary step toward translating the treatment to people with CSNB with an eventual clinical trial.

Reference: Miyadera K, Santana E, Roszak K, et al. Targeting ON-bipolar cells by AAV gene therapy stably reverses LRIT3-congenital stationary night blindness. Proceedings of the National Academy of Sciences. 2022;119(13):e2117038119. doi:10.1073/pnas.2117038119

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Gene Therapy Reverses Night Blindness in Dogs - Technology Networks

Docket Number: FDA-2021-D-0398 Issued by:

Guidance Issuing Office

Center for Biologics Evaluation and Research

In this guidance, we, FDA, are providing recommendations to sponsors developing human gene therapy products incorporating genome editing (GE) of human somatic cells. Specifically, this guidance provides recommendations regarding information that should be provided in an Investigational New Drug (IND) application in order to assess the safety and quality of the investigational GE product, as required in Title 21 of the Code of Federal Regulations 312.23 (21 CFR 312.23). This includes information on product design, product manufacturing, product testing, preclinical safety assessment, and clinical trial design.

You can submit online or written comments on any guidance at any time (see 21 CFR 10.115(g)(5))

If unable to submit comments online, please mail written comments to:

Dockets ManagementFood and Drug Administration5630 Fishers Lane, Rm 1061Rockville, MD 20852

All written comments should be identified with this document's docket number: FDA-2021-D-0398.

03/21/2022

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Human Gene Therapy Products Incorporating Human Genome Editing - FDA.gov

DUBLIN, March 24, 2022 /PRNewswire/ -- The "Cell and Gene Therapy World Market and Market Potential" report has been added to ResearchAndMarkets.com's offering.

The report examines developments in cell and gene therapy markets by condition/disorder, including principal products, trends in research and development, market breakdown of cell and gene therapies, regional market summary, and competitor summary.

Cell and Gene Therapy World Market and Market Potential presents the market in segments that provide an overview of disease epidemiology, market estimates and forecasts, and competitive summary of leading providers:

The following conditions/disorders are covered:

Dermatology, including:

Cardiovascular and Blood Disorders, including:

Oncology, including:

Ophthalmic Conditions, including:

Musculoskeletal Conditions and Disorders, including:

Other Conditions, including:

Key Topics Covered:

CHAPTER 1: EXECUTIVE SUMMARY

CHAPTER 2: INTRODUCTION TO CELL AND GENE THERAPY

CHAPTER 3: CELL AND GENE THERAPY MARKETS IN DERMATOLOGY OVERVIEW

CHAPTER 4: CELL AND GENE THERAPY MARKETS IN CARDIOVASCULAR AND BLOOD DISORDERSCHAPTER 5: CELL AND GENE THERAPY MARKETS IN ONCOLOGY

CHAPTER 6: CELL AND GENE THERAPY MARKETS IN OPHTHALMIC CONDITIONS

CHAPTER 7: CELL AND GENE THERAPY MARKETS IN MUSCULOSKELETAL CONDITIONS AND DISORDERS

CHAPTER 9: CELL AND GENE THERAPY MARKETS IN OTHER CONDITIONS

CHAPTER 10: CELL AND GENE THERAPY MARKET REVIEW

CHAPTER 11: MARKET PARTICIPANTS

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Worldwide Cell and Gene Therapy Industry to 2030 - Players Include Amgen, Biogen and Bluebird Bio Among Others - PR Newswire