Category Archives: Genetic medicine

Genome Editing Market: Snapshot

Genome editing tools have come a long way from the mid-twentieth century. In 1970s and 1980s, gene targeting was done using largely homologous combination, but was only possible in mice. Since then, the expanding science of genetic analysis and manipulation extended to all types of cells and organisms. Advent of new tools helped scientists achieve targeted DNA double-strand break (DSB) in the chromosome, and is a key pivot on which revenue generation in the genome editing market prospered. New directions for programmable genome editing emerged in the decades of the twenty-first century, expanding the arena.

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Cutting-edge platforms at various points in time continue to enrich genome editing market. Various classes of nucleases emerged, most notable of which is CRISPR-Cas. Research labs around the world have extensively used the platforms in making DSBs at any target of choice. Aside from this, agricultural sciences and medical sectors make substantial use of zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) in genome editing. Strides made in stem cell therapies, particularly in rectifying an aberrant mutation, have boosted the growth of the genome editing market. Genetic diseases such as muscular dystrophy and sickle cell disease present an incredible revenue prospect in the genome editing market. Ongoing research on novel vectors and non-vector approaches are expected to bolster the outlook of the market.

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Genomic editing refers to the strategies and techniques implemented for the modification of target genetic information of any living organism. Genome editing involves gene modification at specific areas through recombinant technology, which increases precision in insertion and decreases cell toxicity. Current advancement in genome editing is based on programmable nucleases. The genome editing market is presently witnessing significant growth due to increase in R&D expenditure, rise in government funding for genomic research, technological advancements, and growth in production of genetically modified crops. Companies have made significant investments in R&D in the past few years to develop cutting-edge technologies, such as, CRISPR and TALEN. For instance, Thermo Fisher Scientific is investing significantly in the development of its CRISPR technology for providing better efficiency and accuracy in research and also to fulfil the unmet demands in research and therapeutics. Cas9 protein and FokI protein have been combined to form a dimeric CRISPR/Cas9 RNA-guided FokI nucleases system, which is expected to have wide range of genome editing applications.

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The genome editing market is growing rapidly due to its application in a large number of areas, such as mutation, therapeutics, and agriculture biotechnology. Genome editing techniques offer large opportunities in crop improvement. However, the real potential of homologous recombination for crop improvement in targeted gene replacement therapy is yet to be realized. Homologous recombination is expected to be used as an effective methodology for crop improvement, which is not possible through transgene addition. Rise in the number of diseases and applications is likely to expand the scope of genome editing in the near future. It includes understanding the role of specific genes and processes of organ specific stem cells, such as, neural stem cells and spermatogonial stem cells. Genome editing has a significant scope to treat genetically affected cells, variety of cancers, and agents of infectious diseases such as viruses, bacteria, parasites, etc. However, genetic alteration of human germline for medicinal purpose has been debated for years. Ethical issues, comprising concern for animal welfare, can arise at all stages of generation and life span of genetically engineered animal.

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The global genome editing market can be segmented based on technology, application, end-user, and geography. In terms of technology, the genome editing market can be categorized into CRISPR, TALEN, ZFN, and other technologies. Bioinformatics has eased the process of data analysis through various technological applications. On the basis of application, the global genome editing market can be classified cell-line engineering, animal genome engineering, plant genome engineering, and others. Based on end-user, the genome editing market can be segmented into pharmaceutical and biotechnological companies and academic and clinical research organizations. In terms of region, the global genome editing market can be segmented into North America, Europe, Asia Pacific, Latin America, and Middle East & Africa. North America is projected to continue its dominance in the global genome editing market owing to high government funding for research on genetic modification in the region. Asia Pacific is a rapidly growing genome editing market due to rise in investments by key players in the region. Rise in drug discovery and development activities, coupled with increasing government initiatives toward funding small and start-up companies in the biotechnology and life sciences industry, is a major factor expected to drive the genome editing market in North America during the forecast period. Players should invest in the emerging economies and the countries of Asia-Pacific like China, South Korea, Australia, India and Singapore in which the genome editing market is expected to grow at rapid pace in future, due to growing funding in research.

Key players operating in the global genome editing market are CRISPR Therapeutics, Thermo Fisher Scientific, GenScript Corporation, Merck KgaA, Sangamo Therapeutics, Inc., Horizon Discovery Group, Integrated DNA Technologies, New England Biolabs, OriGene Technologies, Lonza Group, and Editas Medicine.

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- First clinical combination study of BBP-398 and LUMAKRAS set to evaluate safety and preliminary efficacy in solid tumors with the KRAS G12C mutation

PALO ALTO, Calif., Jan. 13, 2022 (GLOBE NEWSWIRE) -- BridgeBio Pharma, Inc. (Nasdaq: BBIO) (BridgeBio), a commercial-stage biopharmaceutical company focused on genetic diseases and cancers, today announced a non-exclusive clinical collaboration with Amgen Inc. (Amgen) to evaluate the combination of BBP-398, a potentially best-in-class SHP2 inhibitor, with LUMAKRAS (sotorasib), a KRASG12C inhibitor, in patients with advanced solid tumors with the KRAS G12C mutation.

The Phase 1/2 study will include a dose escalation period followed by dose expansion and optimization, and is designed to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics and preliminary efficacy of BBP-398 in combination with LUMAKRAS. Under the terms of the non-exclusive collaboration, BridgeBio will sponsor the study and Amgen will provide a global supply of LUMAKRAS.

BBP-398 is a potent small-molecular inhibitor of SHP2 developed in collaboration with The University of Texas MDAnderson Cancer Centers Therapeutics Discovery division. SHP2 is a protein-tyrosine phosphatase that links growth factor, cytokine and integrin signaling with the downstream RAS/ERK MAPK pathway to regulate cellular proliferation and survival. By combining SHP2 inhibition with KRASG12C inhibition in patients with the KRAS G12C mutation, there is potential that the investigational combination could prevent oncogenesis and overactive cellular proliferation.

Overactivity of the MAPK pathway is a significant cause of many types of difficult-to-treat cancers and by combining these two agents, we aim to reduce the oncogenic potential of tumor cells, said Frank McCormick, Ph.D., chairman of oncology at BridgeBio. Building on our collaborations with Bristol Myers Squibb and LianBio, we are excited to be working with Amgen on this new collaboration. By harnessing the power of BBP-398 as a potentially best-in-class SHP2 inhibitor with LUMAKRAS, we are hopeful that we will be able to provide substantial relief for cancer patients in need. We will continue to pursue additional collaborations that we believe hold promise for patients.

KRAS mutations occur in approximately 17% of malignant solid tumors. BBP-398, as a monotherapy or in combination with other targeted therapies, could potentially be a promising therapy for patients with the KRAS G12C mutation.

BridgeBio is currently advancing its Phase 1 clinical trial of its SHP2 inhibitor, BBP-398, in patients with solid tumors driven by mutations in the MAPK signaling pathway, including RAS and receptor tyrosine kinase genes. BBP-398 is part of BridgeBios growing precision oncology pipeline and is one of 14 programs in the broader portfolio that are being advanced in the clinic or commercial setting.

About BBP-398BBP-398 is a potentially best-in-class SHP2 inhibitor. Earlier this year, BridgeBio entered a non-exclusive, co-funded clinical collaboration with Bristol Myers Squibb to evaluate the combination of BBP-398 with OPDIVO (nivolumab) in patients with advanced solid tumors with KRAS mutations. BridgeBio previously also entered into a strategic collaboration with LianBio for clinical development and commercialization of BBP-398 in combination with various agents in solid tumors such as non-small cell lung cancer, colorectal cancer and pancreatic cancer in mainland China and other major Asian markets.

About BridgeBio Pharma, Inc.BridgeBio Pharma (BridgeBio) is a biopharmaceutical company founded to discover, create, test and deliver transformative medicines to treat patients who suffer from genetic diseases and cancers with clear genetic drivers. BridgeBios pipeline of over 30 development programs ranges from early science to advanced clinical trials and its commercial organization is focused on delivering the companys two approved therapies. BridgeBio was founded in 2015 and its team of experienced drug discoverers, developers and innovators are committed to applying advances in genetic medicine to help patients as quickly as possible. For more information visitbridgebio.comand follow us onLinkedInandTwitter.

BridgeBio Pharma, Inc. Forward-Looking StatementsThis press release contains forward-looking statements. Statements we make in this press release may include statements that are not historical facts and are considered forward-looking within the meaning of Section 27A of the Securities Act of 1933, as amended (the Securities Act), and Section 21E of the Securities Exchange Act of 1934, as amended (the Exchange Act), which are usually identified by the use of words such as anticipates, believes, estimates, expects, intends, may, plans, projects, seeks, should, will, and variations of such words or similar expressions. We intend these forward-looking statements to be covered by the safe harbor provisions for forward-looking statements contained in Section 27A of the Securities Act and Section 21E of the Exchange Act and are making this statement for purposes of complying with those safe harbor provisions. These forward-looking statements, including statements relating to expectations, plans, and prospects regarding the success of our non-exclusive clinical collaboration with Amgen, the timing and success of a Phase 1/2 study to evaluate the safety and preliminary efficacy of BBP-398 in combination with LUMAKRAS in patients with advanced solid tumors with the KRAS G12C mutation, the ability of combining SHP2 inhibition with KRASG12C inhibition in patients with the KRAS G12C mutation to prevent oncogenesis and overactive cellular proliferation, our ability to provide substantial relief for cancer patients in need, the promise of targeted therapies for patients with KRAS mutations, the success and status of current and future relationships with third-party collaborators and academic partners, the continuing success of our clinical collaboration with Bristol Myers Squibb to evaluate the combination of BBP-398 with OPDIVO (nivolumab), and the potential ability of our product candidates to treat genetically driven diseases and cancers with clear genetic drivers, reflect our current views about our plans, intentions, expectations, strategies and prospects, and are based on the information currently available to us and on assumptions we have made and are not forecasts, promises nor guarantees. Although we believe that our plans, intentions, expectations, strategies and prospects as reflected in or suggested by these forward-looking statements are reasonable, we can give no assurance that the plans, intentions, expectations or strategies will be attained or achieved. Furthermore, actual results may differ materially from those described in the forward-looking statements and will be affected by a number of risks, uncertainties and assumptions, including, but not limited to, the success of our product candidates to treat genetically driven diseases and cancers with clear genetic drivers, the continuing success of our collaboration with Amgen and other third parties, our ability to enter into future collaboration agreements, potential adverse impacts due to the global COVID-19 pandemic such as delays in regulatory review, manufacturing and clinical trials, supply chain interruptions, adverse effects on healthcare systems and disruption of the global economy, as well as those risks set forth in the Risk Factors section of BridgeBios most recent Annual Report on Form 10-K and BridgeBios other SEC filings. Moreover, we operate in a very competitive and rapidly changing environment in which new risks emerge from time to time. Except as required by applicable law, we assume no obligation to update publicly any forward-looking statements, whether as a result of new information, future events or otherwise.

BridgeBio Media Contact:Grace RauhGrace.rauh@bridgebio.com(917) 232-5478

BridgeBio Investor Contact:Katherine Yaukatherine.yau@bridgebio.com(516) 554-5989

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BridgeBio Announces Clinical Collaboration with Amgen to Study BBP-398, a Potentially Best-in-class SHP2 Inhibitor, in Combination with LUMAKRAS...

Significance

Functionalization of proteins and biopolymers with chemical modifications can be utilized to alter their chemical and biophysical properties. In contrast to traditional chemical functionalization strategies, the use of nonstandard amino acids enables precise positioning of functional groups. Here, we report that multisite conjugation of fatty acids, at precise sites harboring genetically encoded nonstandard amino acids with bioorthogonal chemical handles, can be employed to tune the half-life of proteins in a mouse model. This programmable approach could offer a technical foundation for the modification of protein and peptide therapeutics to improve their efficacy or pharmacokinetic profile (e.g., to prevent rapid clearance and reduce frequency of administration).

The use of biologics in the treatment of numerous diseases has increased steadily over the past decade due to their high specificities, low toxicity, and limited side effects. Despite this success, peptide- and protein-based drugs are limited by short half-lives and immunogenicity. To address these challenges, we use a genomically recoded organism to produce genetically encoded elastin-like polypeptideprotein fusions containing multiple instances of para-azidophenylalanine (pAzF). Precise lipidation of these pAzF residues generated a set of sequence-defined synthetic biopolymers with programmable binding affinity to albumin without ablating the activity of model fusion proteins, and with tunable blood serum half-lives spanning 5 to 94% of albumins half-life in a mouse model. Our findings present a proof of concept for the use of genetically encoded bioorthogonal conjugation sites for multisite lipidation to tune protein stability in mouse serum. This work establishes a programmable approach to extend and tune the half-life of protein or peptide therapeutics and a technical foundation to produce functionalized biopolymers endowed with programmable chemical and biophysical properties with broad applications in medicine, materials science, and biotechnology.

A major goal of synthetic biology is to harness biological systems to produce valuable products, such as new therapeutics, renewable chemicals, and functionalized materials. In the case of proteins, the native translation process uses information encoded in DNA to guide their template-directed production at monomeric precision, albeit limited to the chemistry of the 20 natural amino acids. Work in genetic code expansion with nonstandard amino acids (nsAAs) has expanded the chemical palette of biology through the template-directed biosynthesis of proteins with synthetic chemistries (1). To date, such work has been limited to only one or a few instances of site-specific incorporation of nsAAs per protein, constraining biopolymer synthesis to tag-and-modify approaches or simple protein decorations. Recent advances include an increase in the number and chemical diversity of nsAAs (1), the development of highly active translation machinery for efficient incorporation of nsAAs into proteins (2, 3), and engineered strains of Escherichia coli with recoded genomes possessing open coding channels that can be dedicated to the incorporation of nsAAs (46). Together, these advances enable multisite incorporation of nsAAs to endow proteins and sequence-defined biopolymers with new chemical and biophysical properties.

An active area of interest for the use of nsAAs is to enhance the functionality of protein and peptide pharmaceuticals. They represent a versatile and fast-growing class of biological therapeutics (7, 8) that are particularly attractive as potential pharmaceuticals due to their high specificity, high activity and, in the case of peptides, rapid tissue penetration (7). However, major barriers prevent the widespread clinical use of many peptide or protein-based therapeutics (7): 1) the need to administer them by injection, 2) their rapid clearance by the kidneys, and 3) their rapid proteolytic degradation. As a result, these pharmaceuticals must be frequently administered at high doses, leading to a peak-and-valley pharmacokinetic profile. These characteristics can negatively affect therapeutic efficacy, can cause undesirable side effects with reduced patient compliance (912), and can trigger an immune response, including the induction of a neutralizing antibody response (13, 14).

To address these challenges, proteins and peptides are frequently functionalized to extend their half-life and improve immunotolerance. A widely adopted strategy is the conjugation of poly(ethylene glycol) (PEG), which increases the radius of the protein and reduces proteolytic cleavage, and consequently reduces clearance (15). However, the development of alternatives to PEGylation remains important, as PEGylation does not always offer the desired effect on pharmacokinetics, and in certain cases, safety concerns about its immunogenicity and accumulation in tissues have been raised (1618). An alternative strategy is to conjugate or fuse the therapeutic protein or peptide to serum proteins with long half-lives, such as serum albumin, antibodies (e.g., full-length or fragments of IgG), or blood components, such as red blood cells (17, 18). Similarly, many approaches use chemical moieties or peptides to promote noncovalent binding interactions to the same serum proteins and complexes in order to extend half-life (1720). One effective and safe option is the use of fatty acids (FAs) to promote binding to serum albumin. For example, insulin and glucagon-like peptide-1 (GLP-1) conjugated with a single FA are clinically used to treat diabetic patients (2123).

A major hurdle to the development of functionalized therapeutics is to selectively and predictably modify the protein while maintaining bioactivity. Conventional strategies for PEGylation and functionalization with chemical moieties utilize chemistries that modify the target protein at their termini, or at residues with reactive side-chains (24). The functionalization at C or N termini can be highly selective and predictable, but it can reduce bioactivity and is thus incompatible with many proteins. In contrast, modifications at reactive side-chains (e.g., cysteine or lysine) is less restrictive, but it can be difficult (or practically impossible) to identify unique reactive sites in the peptide sequence for site-specific conjugation. To address this problem, amino acids in the protein are typically mutagenized, which can result in reduced bioactivity. Recently, nsAAs have been successfully employed for modification of proteins and peptides, offering bioorthogonal chemistries for functionalization at predetermined positions within the protein (2426). For example, human growth hormone (hGH) and fibroblast growth factor 21 (FGF21) were site-specifically PEGylated, prolonging their function through extended serum half-life in clinical trials (27, 28). In other cases, site-specific lipidation at a single nsAA was shown to extend half-life in mouse models (29, 30). Although these approaches have improved protein half-life, typically these designs are constrained to a single instance of the nsAA, which limits the versatility and tunability (e.g., customized range of half-lives) of these functionalized peptides and proteins.

In this study, we present a synthetic biology platform to biosynthesize proteinpolymer fusions with sequence-defined conjugation sites for multisite lipidation in order to extend and tailor the half-life of proteins invivo. Specifically, we encoded up to 10 instances of the nsAA para-azidophenylalanine (pAzF) in elastin-like polypeptide (ELP) fusion proteins at high yields in an engineered bacterium with a recoded genome (4), and demonstrate the ability to precisely control the position and number of FAs per biopolymer. We found that the number of FAs per protein is strongly correlated with the binding affinity to serum albumin, enabling us to tune the invivo serum half-life of proteins without accumulation in organs or eliciting an inflammatory response in mouse. These advances could be applied to extend and tune the half-life of protein or peptide therapeutics and establish a technical foundation to produce sequence-defined programmable biopolymers endowed with bespoke chemical and biophysical properties with broad applications in medicine, materials science, and biotechnology.

To enable the biosynthesis of sequence-defined synthetic biopolymers with template-directed conjugation sites, we utilized a recently described synthetic biology expression system that allows efficient incorporation of nsAAs (e.g., pAzF) at UAG codons (Fig. 1A) (2). This system possesses two unique properties. First, the expression host is the genomically recoded organism (GRO) (4), an E. coli MG1655 derivative, in which all instances of UAG stop codons were recoded to synonymous UAA codons, followed by the deletion of release factor 1 (RF1). This GRO establishes an open codon by eliminating competition between an orthogonal tRNACUA/aminoacyl tRNA synthase (aaRS) pair and termination at UAG codons by RF1. Second, aaRSs evolved for aminoacylation with nsAAs typically have significantly reduced activities compared to native enzymes, resulting in low levels of nsAA-tRNA and low yields for proteins with multiple instances of an nsAA (31). Here, we use a tRNACUA/aaRS derived from Methanocaldococcus jannaschii that was evolved for enhanced activity, enabling efficient multisite incorporation of nsAAs into proteins (2). Together, this expression system enables the biosynthesis of polypeptide polymers with multiple pAzF residues at high yields and accuracy.

Biosynthesis and functionalization of genetically encoded biopolymers for half-life extension of proteins. (A) Site-specific multisite incorporation of pAzF at UAG codons in the GRO. All 321 TAG codons in E. coli were genomically recoded to TAA. To create the GRO, RF1 was deleted. The canonical amino acids and pAzF are shown as black and red circles, respectively. The TAG codon is converted into a sense codon for multisite incorporation of pAzF. (B) Schematic of the ELP-protein with 10 pAzF residues. The chemical structure of pAzF and the sequence of a single ELP repeat are shown. (C) Functionalization of azido groups in ELPs through copper(I)-mediated click chemistry with alkynyl palmitic acid. (D) Functionalized biopolymers are characterized in mice to study impact on half-life.

To study the effect of the number of FA conjugates on the invivo serum half-life, we chose to introduce the nsAA in an ELP with 10 consecutive pentadecapeptide repeats for functionalization. ELPs have previously been fused to active therapeutic peptides for a variety of indications, including type 2 diabetes and cancer (32), and serve as a versatile module to alter their pharmacokinetics. Within each repeat of the ELP, we encoded either a tyrosine or a pAzF residue at a designated guest residue position (henceforth named the target residue) (Fig. 1B), such that the genetic template controls the number and position of pAzF residues in the ELP-GFP. In turn, the bioorthogonal copper(I)-catalyzed azide-alkyne Huisgen cycloaddition (click-chemistry) reaction between pAzF residues and palmitic acid alkynes (with an terminal alkyne, such that the carboxyl group is exposed) ensures site-specific conjugation (Fig. 1 C and D). Alkynyl palmitic acid was used for functionalization because it had previously been shown to strongly promote binding to albumin (23).

To evaluate if we could produce proteins with a genetically controlled number of FAs, we expressed ELP-GFP with 0, 1, 5, or 10 UAG codons at a yield of 70 mg/L (Fig. 2A and SI Appendix, Fig. S1). To carefully examine the fidelity and efficiency of each step in our system, we performed quantitative mass spectrometry (MS) analysis of the ELPs digested with thermolysin, which liberates each of the 10 constituent ELP units. To account for differences in ionization efficiency between the different peptide species, ion counts were quantified using a standard curve for each peptide (SI Appendix, Fig. S2). We first evaluated the efficiency of pAzF incorporation and found that the abundance of ELP units with pAzF was directly proportional to the number of UAG codons in the construct (SI Appendix, Fig. S3). Consistent with prior work (2), when all 10 ELP units contained a UAG codon, we detected minor (<5%) tyrosine misincorporation.

Analysis of the purity and bioactivity of multisite lipidated biopolymers. (A) Schematic representation of ELP-GFP reporter constructs with 1, 5, or 10 pAzF residues. Target positions for pAzF are indicated in red. (B) Relative abundance of detected nonstandard amino acids at target residues of ELP units based on quantitative MS. Treatment with ISAz selectively converts reduced pAzF residues, pAF, back into pAzF. (C) Click-chemistry with FA alkynes functionalizes all pAzF, but not pAF, residues (n = 3, error bars: mean SD). (DG) Intact MS of full-length ELP(FA)-GFP after click-chemistry with (blue) or without (black) ISAz treatment. (H) Activity of recombinant trx, trx-ELP(10pAzF), and trx-ELP(10FA) at protein quantities ranging from 25 to 150 pmol per well. (I) Effect of HSA on the activity of recombinant trx, trx-ELP(10pAzF) and trx-ELP(10FA) using 100 pmol of each protein and 500 pmol HSA. Data are normalized to the activity of each protein without HSA (n = 3, error bars: mean SEM). *P < 0.05, **P < 0.01.

While examining the fidelity of pAzF incorporation, we observed significant levels of para-aminophenylalanine (pAF) (Fig. 2B), the reduced form of pAzF, which cannot participate in click-chemistry. In our system, pAF is the result of pAzF reduction (2, 33), and causes significant impurities and heterogeneity in the final preparations if left unresolved. To overcome this impurity, we developed a method to selectively recover pAzF from pAF with the diazotransfer reagent imidazole-1-sulfonyl azide (ISAz) (Materials and Methods) (34). We previously demonstrated that this approach enabled effective conversion of pAF residues to pAzF, without introducing azides at other primary amines found in ELP-GFPs (34). Consistent with this prior work, after treatment with ISAz we observed less than 5% of pAF via quantitative MS in each of the digested ELP-GFP constructs (Fig. 2B).

We then used click-chemistry to attach alkynyl palmitic acid at the precise positions where pAzF was encoded and assessed the purity of each ELP-GFP construct. These functionalized constructs are denoted as ELP(nFA)-GFP, where n indicates the number of UAG codons encoding pAzF in the template. We observed that all pAzF residues were converted to FA conjugates and no further reduction to pAF was detected during this reaction (Fig. 2C), emphasizing the high efficiency of this conjugation strategy. To complement the quantification at the peptide level, we used MS of the intact protein to evaluate the purity of the products (Materials and Methods). We consistently observed one dominant peak at the expected mass after ISAz treatment, whereas untreated samples demonstrated heterogeneous modification of the ELP-GFP (Fig. 2 DG). For example, the ELP(10FA)-GFP without ISAz treatment showed multiple distinct peaks corresponding to an impure biopolymer with variable number of FAs. The peak profile correlates with a binomial distribution determined by the availability of pAzF residues (SI Appendix, Fig. S4) and suggests pAzF reduction is probabilistic.

Finally, we evaluated if the addition of multiple FAs per protein would impair solubility of the resulting constructs. The solubility of unmodified ELP-GFP and ISAz-treated proteins with 1, 5, or 10 FAs were determined by dynamic light scattering (DLS) analysis. All constructs, before and after FA conjugation, were soluble (>99% by volume) and did not self-assemble in solution (SI Appendix, Fig. S5). Together, these results demonstrate that the genetically controlled placement of pAzF and chemical regeneration of reduced pAF residues enable the programmable and robust functionalization of biopolymers at multiple sites.

We evaluated the effect of ELP and FA conjugation of peptide bioactivity on the activity of two proteins. We first quantified the effect of FA conjugation on GFP fluorescence, and found that it is reduced by 14 to 25% (SI Appendix, Fig. S6). In addition, to examine the effect of ELP fusion and FA conjugation on enzymatic activity, we produced and characterized thioredoxin (trx)-ELP fusion proteins. We evaluated the activity of recombinantly produced trx, trx-ELP(10pAzF), and trx-ELP(10FA), after ISAz treatment. Similar bioactivity was observed for trx and trx-ELP(10pAzF), while trx-ELP(10FA) retained greater than 50% bioactivity (Fig. 2H). In addition, we evaluated the effect of human serum albumin (HSA) binding on the activity of these proteins. To this end, we performed an activity assay in the presence of fivefold excess HSA concentration, and found that HSA binding reduced the activity of trx-ELP(10FA) by 50%, while activity of trx and trx-ELP(10pAzF) were not significantly reduced (Fig. 2I). Although FA conjugation and HSA binding may partially reduce bioactivity, these analyses demonstrate that posttranslational functionalization with FAs is compatible with bioactive proteins.

Since prior work with single FA conjugations of insulin showed that serum half-life is correlated with the binding affinity to albumin (23), we hypothesized that multisite lipidation of ELP-GFPs would significantly enhance binding affinity to mouse serum albumin (MSA), and consequently extend serum half-life, compared to a single conjugated FA. To study the impact of increasing the number of FAs, we analyzed ELP-GFP constructs (both with and without ISAz treatment) with surface plasmon resonance (SPR). The KD values of our constructs were estimated based on the steady-state binding (Table 1). There was no detectable binding between MSA and the negative control without conjugated FAs [ELP(0FA)-GFP]. For untreated biopolymers, we found that the KD with a single FA, ELP(1FA)-GFP (KD = 126 32 M), was lowered 12- to 45-fold for ELP(5FA)-GFP (KD = 10.4 4.0 M) and ELP(10FA)-GFP (KD = 2.8 0.2 M), respectively. For the ISAz-treated biopolymers, we observed much stronger binding overall: treated ELP(1FA)-GFP presented a KD of 25.9 7.1 M, and an increase to 5 and 10 FAs per protein further lowered the KD to 4.0 1.6 M and 2.22 0.03 M, respectively. These data indicate that the affinity for MSA is strongly enhanced by conjugation of multiple FAs per protein and confirm that the binding affinity is correlated with the number of FAs.

Binding affinity of ELP-GFP constructs for serum albumin

We next determined if the tighter binding affinity is translated to prolonged half-life in C57BL/6J mice. A total of 50 g of each protein variant (10 M in phosphate-buffered saline [PBS]) was injected intravenously and blood was collected after 1, 4, 8, 16, and 24 h, followed by daily collections for 7 d. The blood levels of ELP(nFA)-GFP constructs were measured using a GFP-specific ELISA, and their pharmacokinetic profiles were calculated (SI Appendix, Fig. S7 and Table S1). We observed a striking 16- to 19-fold increase in half-life from 1.7 h for ELP(0FA)-GFP to 28 to 33 h for ISAz-treated ELP(5FA)-GFP and ELP(10FA)-GFP, as well as for untreated ELP(10FA)-GFP (Fig. 3A). Notably, when the same constructs were injected subcutaneously, we observed a delayed peak concentration, but the half-lives were equivalent to intravenous injections (SI Appendix, Figs. S8 and S9). Furthermore, these data show that the half-life of tight binding ELP-GFP constructs with multiple FA conjugates approaches the half-life of MSA in mice (35 h) (35), and is similar to the half-life of 28 h reported for proteinMSA fusion proteins (36).

In vivo characterization of lipidated biopolymers in mouse. (A) Serum half-life measurements of lipidated biopolymers with or without treatment with ISAz. Measurements were collected after a single intravenous injection of 50 g biopolymer in C57BL/6J mice. ELP(0FA)-GFP after ISAz treatment was not measured (n = 4, error bars: mean SD). (B) Correlation between the KD and half-life of lipidated biopolymers with or without ISAz treatment. The horizontal, black dotted line shows the half-life of MSA, the dashed gray line shows model predictions (n = 4 to 8, error bars: mean SD, n.d. = not detected). (C) Distribution of biopolymers in mouse organs, 3 and 48 h after intravenous injection of Alexa Fluor 648labeled ELP(0FA)-GFP or ELP(10FA)-GFP. The biopolymers were treated with ISAz. The data are representative of four independent measurements. (D) Quantification of average Alexa Fluor 648 intensity for organs shown in C (n = 4, error bars: mean SEM). For each organ separately, a one-way ANOVA was used to determine whether the differences between the means of the five treatment groups were statistically significant. After multiple testing correction, *P < 0.05 and **P < 0.005. (E) Serum concentration of select inflammatory cytokines at 3 and 48 h after injection. Endotoxin (100 g) and PBS were used as positive and negative controls, respectively. Measurements below the lower limit of detection (20 pg/mL) or above the upper limit of detection (5,000 pg/mL) are plotted at their limit of detection (n = 3, error bars: mean SD).

Finally, we note that the observed KD values can be approximated by dividing the KD of ELP(1FA)-GFP (ISAz-treated) by the average number of conjugated FAs (SI Appendix, Fig. S10). This correlation has two key implications. First, biopolymers with high numbers of FA conjugates [i.e., ISAz-treated ELP(5FA)-GFP, and both treated and untreated ELP(10FA)-GFP] will have similar binding affinities to albumin, and therefore also similar half-lives. Second, tuning of the binding affinity and half-life is most pronounced at lower numbers of conjugated FAs. In the case of ISAz-treated ELP(10FA)-GFP, we found a small decrease (although not statistically significant) in half-life compared to treated ELP(5FA)-GFP and untreated ELP(10FA)-GFP. We hypothesize that denser packing of the 10 FAs does not improve, or may even reduce, the availability of FAs for albumin binding, highlighting the value of being able to precisely control the number of FAs per protein.

We computationally modeled the system to gain a deeper understanding of the correlation between the binding affinity and half-life (SI Appendix, Supplementary Information Text and Fig. S11). In brief, a set of ordinary differential equations describes the binding and release of ELP-GFP from albumin as a function of the KD, as well as the clearance of both bound and unbound ELP-GFP. Here, unbound ELP-GFP has a half-life of 1.7 h, as empirically determined, and bound ELP-GFP is cleared at the same rate as albumin (35 h for mice) (35). Importantly, the half-life of the protein is determined by three parameters in this model: 1) the half-life of unbound protein, 2) the half-life of serum albumin, and 3) the binding affinity between the protein and albumin. By simulating the kinetics over time, we were able to calculate the overall clearance rate, and predictions made by the model were in good agreement with the empirical measurements for KD and half-life (Fig. 3B and SI Appendix, Fig. S11). This suggests predictive capability for the half-life based on empirically determined KD values, or the model can provide a target KD based on the desired half-life. Together, our results confirm that titrating the number of FAs allows predictable tuning of the protein half-life by modifying the binding affinity to albumin.

To evaluate the biocompatibility of the ELP-GFP constructs, we assessed the biodistribution and inflammatory response in mouse. ELP(0FA)-GFP and ELP(10FA)-GFP labeled with Alexa Fluor 647 dye were administered intravenously, and after 3 or 48 h, the brain, lungs, heart, spleen, liver, kidneys, and blood were collected and imaged for far-red fluorescence (Fig. 3 C and D and SI Appendix, Fig. S12). In the case of ELP(0FA)-GFP, most of the reporter had cleared from the blood after 3 h, and a strong signal was observed in the kidney, whereas ELP(10FA)-GFP was clearly observed in the blood, and to a lesser extent in the kidney. For both samples, a small increase (20 to 30%) in signal was observed in the liver. After 48 h, the Alexa Fluor 647 signal was only observed in the blood of ELP(10FA)-GFPinjected mice, whereas the intensity in all other organs had returned to the basal level seen in the PBS injection control. These results are consistent with the rapid clearance of ELP(0FA)-GFP, which is likely to occur mostly through excretion from the kidneys. The blood from each of these conditions was further analyzed for signs of inflammation. We did not detect any elevation of proinflammatory cytokine levels after injection of ELP-GFP constructs compared to PBS injection, whereas injection with lipopolysaccharide (LPS) as positive control gave a clear inflammatory response at both 3 and 48 h (Fig. 3E). Together, these results show that FA conjugation enables half-life extension without long-term accumulation in organs, or eliciting an inflammatory response after intravenous injection.

Considering applications of this technology for peptide and protein drug delivery in humans, we evaluated if the use of multiple FAs per protein conveyed similar increases in binding affinity to HSA. We observed KD values of 19.3 3.9 M, 3.2 0.6 M, and 1.6 0.2 M for ELP-GFP constructs with 1, 5, and 10 FAs per protein, respectively (Table 1). These binding affinities closely mirror the values observed for MSA, suggesting that multisite lipidation of proteins could be a promising strategy to tailor protein half-life in humans.

In this study, we describe the design and production of sequenced-defined synthetic biopolymers conjugated with a programmable number of FAs to tailor the serum half-life of proteins. Specifically, the genetically encoded pAzF residues facilitate precise and programmable functionalization with FAs, which enables titration of the binding affinity to both MSA and HSA. We determined that the binding affinity to albumin was predictive of the serum half-life in mice, suggesting that the protein clearance can be tuned by controlling the number of conjugated FAs per protein. Notably, we measured serum half-lives of up to 33 h, which is 94% of the 35-h half-life of MSA. Importantly, with similar binding affinities for MSA and HSA, we hypothesize that the half-life of these same constructs will be higher in humans, given that HSA has a significantly longer half-life (19 d) (37). Furthermore, activity analysis of a trx fusion protein, trx-ELP(10FA), indicate at most a 50% activity loss (in the absence or presence of HSA), as compared with free trx. This compares favorably with other carriers reported to cause an 30- to 500-fold reduction in the activity of other peptides (3842). Although, as is true for any carrier, the effect of ELP fusion and FA conjugation on activity is expected to vary for each individual peptide, protein, or molecule, our proposed fusion partner is highly tunable, in ELP size, sequence, and FA number and position, which should enable future optimization of both pharmacokinetics and bioactivity of each drug candidate.

Lipidation is an appealing alternative to PEG, which has come under scrutiny due to concerns about immunogenicity (43, 44), and uncertainty about its degradation and clearance from the body (45). The use of FAs has clinical precedence, offers greater tunability than direct fusion to albumin, and has a well-established safety profile (46). However, the utility of current lipidation strategies is constrained by two factors. First, typically only moderate half-life extensions are achieved due to weak binding of pharmaceuticals with single FAs to albumin. Second, the ability to identify uniquely reactive residues without impacting bioactivity remains challenging with conventional labeling strategies. Our work addresses both limitations with a general methodology that enables tuning the half-life extension by titrating the number of FAs per protein, and the ability to design conjugation sites at monomeric precision enables facile screening of permissive residues to maintain bioactivity.

Unique to this work is the multisite and programmable placement of nsAAs to produce a biopolymer with tunable properties, enabled by sequence-defined insertion of multiple FAs per biopolymer for functionalization. The ELP can be placed at either of the termini, or pAzF residues can be positioned in the primary sequence of the protein, permitting the optimization of both bioactivity and half-life extension, which highlights the flexibility of this approach. Furthermore, bioorthogonal conjugation sites, such as pAzF residues, allow the attachment of a wide variety of chemical moieties to expand the palette of biological chemistry far beyond FAs at genetically encoded positions throughout the protein to enhance its functionality. This establishes a foundation for a new class of synthetic, sequence-defined biopolymers comprised of a combination of natural and synthetic monomers that unites the diversity of the chemical world with the monomeric precision of translation in biological systems. These biopolymers are uniquely enabled by recoded organisms with open coding channels dedicated to the template-directed incorporation of synthetic monomers. We envision that this work, together with further recoding efforts to open up additional coding channels dedicated for multiple distinct nsAAs (46), establishes the basis for novel and programmable biopolymers (47, 48) with broad utility in biological research, pharmaceuticals, materials science, and biotechnology.

All proteins were expressed in the GRO (E. coli C321.A, CP006698.1, GI:54981157) (4) containing a previously described OTS plasmid pAcFRS.1.t1 with a p15A origin of replication and chloramphenicol acetyltransferase selection marker (2). The ELP-GFP genes were expressed from a plasmid with colE1 origin of replication and a kanamycin resistance marker (SI Appendix, Fig. S13). Each ELP-GFP construct had 10 repetitive units of 15 amino acids (VPGAGVPGXGVPGGG), where residue X is either tyrosine or pAzF (SI Appendix, Tables S2 and S3). The gene for trx was chemically synthesized (IDT) and cloned using EcoRI and PpuMI restriction enzymes into an expression vector containing to create trx-ELP(10UAG) (SI Appendix, Table S4). The same trx gene was cloned into an empty-expression vector for expression of unfused trx.

All cultures were grown at 34C under shaking (220 rpm). Before expression, the expression strains were grown to confluence in 50 mL 2xYT media. This culture was used to inoculate 1 liter of 2xYT, containing 30 g/mL chloramphenicol, 20 g/mL kanamycin, 0.2% arabinose, and 1 mM pAzF. After 4 h, expression of ELP-GFP was induced, using a final concentration of 60 ng/mL anhydrotetracycline. Cells were harvested 24 h after inoculation by centrifugation at 4,000 g for 15 min at 4C.

The cell pellet was resuspended in PBS, pH 7.4, and lysed by sonication (12 cycles of 10-s sonication separated by 40-s intervals, 40% amplitude). Poly(ethyleneimine) was added to each lysed suspension to a final concentration of 1.25%, after which the soluble fraction was separated from the cell debris by 15 min of centrifugation at 4,000 g. ELP-GFP proteins were then purified by phase transition triggered by sodium citrate, followed by centrifugation at 15,000 g for 3 min to eliminate contaminant proteins that did not precipitate. Finally, native E. coli proteins were denatured at 75C, and removed by centrifugation. After three purification cycles, the ELP-GFP proteins to >95% purity as judged by Coomassie staining of SDS/PAGE gels.

When stated, pAF residues from ELP-GFP proteins were regenerated using ISAz, as previously described. In brief, diazotransfer reactions were performed for proteins at a concentration of 20 M using 200 equivalents of ISAz in 10 PBS (1.4 M NaCl, 0.1 M phosphate, 0.03 M KCl) pH 7.2 at room temperature. After 72 h, reactions were stopped by exchanging the buffer to PBS (1, pH7.4).

The ELP-GFP proteins were reacted with palmitic acid alkyne using copper(I)-catalyzed azide-alkyne Huisgen cycloaddition (click-chemistry). For this reaction, proteins were diluted to a final azide concentration of 30 M, 35% DMSO, 0.16 mM palmitic acid alkyne, 0.1 mM CuSO4 and 0.5 mM THPTA (premixed for 30 min), 5 mM aminoguanidine hydrochloride, and 5 mM sodium ascorbate. The click-chemistry reaction was incubated for 1 h at room temperature under constant, gentle mixing. After the reaction, the protein was buffer exchanged to PBS (pH 7.4) using Amicon filters (10 kDa molecular weight cutoff [MWCO]).

Proteins for biodistribution studies were further labeled at primary amines with an Alexa Fluor 647 succinimidyl ester. Proteins were diluted to 0.1 mg/mL, and mixed with 5 g/mL fluorophore in PBS for mild labeling. Excess dye was removed using Amicon filters (10 kDa MWCO).

Endotoxins were removed from all protein preparations used for animal experiments, using Pierce high-capacity endotoxin removal columns following the manufacturers protocol (Thermo Fisher Scientific, catalog # 88274). Prior to injection, endotoxin levels were confirmed to be under 0.1 endotoxin unit (EU) per injection using Gel-Clot LAL reagent with sensitivity of 0.06 EU/mL (Charles River, catalog #R12006).

The purity at the target residue was determined by quantitative MS. The ELP-GFP proteins were buffer exchanged and diluted to 15 M in digestion buffer (50 mM Tris, pH 8.0, and 0.5 mM CaCl2), and were digested with 1.5 M thermolysin (Promega) for 6 h at 80C. The resulting ELP-peptides were quantified using standard curves based on synthetic peptides (SI Appendix, Fig. S2). High-resolution MS data were collected using an Agilent iFunnel 6550 quadrupole time-of-flight (TOF) MS with an electrospray ionization (ESI) source, coupled to an Agilent Infinity 1290 ultrahigh-performance liquid chromatography system with an Agilent Eclipse Plus C18 1.8 m, 4.6 50-mm column. Solvents used were (solvent A) water 0.1% formic acid and (solvent B) CH3CN 0.1% formic acid. Mass spectra were gathered using Dual Agilent Jet Stream ESI in positive mode. The mass range was set from 110 to 1,700 m/z with a scan speed of three scans per second. The capillary and nozzle voltages were set to 5,500 and 2,000 V, respectively. The source parameters were set with a gas temperature of 280C and a flowrate of 11 liters/min, nebulizer at 40 psig, and sheath gas temperature at 350C at a flow of 11 liters/min. MS data were acquired with MassHunter Workstation Data Acquisition (version B.06.01, Agilent Technologies) and analyzed using MassHunterQualitative Analysis (version B.07.00, Agilent Technologies).

For MALDI-TOF analysis, 2 L of the protein samples were mixed in a ratio of 1:1:1 with 2% trifluoroacetic acid solution and then with the matrix solution (375 L of 20 mg/mL solution of 2,5-DHAP [2,5-dihydroxy acetophenone] in ethanol and 125 L of 18 mg/mL of aqueous DAC [diammonium hydrogen citrate solution]) by pipetting, until crystallization of the mixture. Then 0.5 L of the protein sample was loaded on MALDI steel target plate and analyzed after solvent evaporation.

MALDI-TOF MS spectra were acquired using an MALDI-TOF/TOF autoflex speed mass spectrometer (Bruker Daltonik), equipped with a smartbeam-II solid-state laser (modified Nd:YAG laser, = 355 nm), at the Ilse Katz Institute for Nanoscale Science and Technology (Ben-Gurion University of Negev, Beer-Sheva, Israel). The instrument was operated in positive ion, linear mode within a mass range from m/z 10 kDa to 50 kDa. Laser fluence were optimized for each sample. The laser was fired at a frequency of 1 kHz and spectra were accumulated in multiples of 500 laser shots, with 1,500 shots in total. Calibration was performed using protein calibration standard from Bruker. Spectrum analysis was performed by the Flexanalysis software.

Protein solubility and self-assembly was analyzed using a Zetasizer Nano ZS (Malvern Pananalytical). For each sample, 11 to 15 acquisitions (determined automatically by the instrument) were obtained at 25C for 10 M protein solutions in PBS. Three separately prepared samples were analyzed, and the analysis for each sample was repeated three times. Populations comprising less than 1% of the total mass (by volume) were excluded from the analysis.

Binding assays were performed on a Biacore T200 instrument. HSA (Sigma, catalog #A3782) or albumin from mouse serum (Sigma, catalog #A3139) were immobilized by amine coupling to research grade CM5 chip (GE Healthcare, catalog #BR100530) from 20 g/mL solutions in 10 mM acetate pH 5.0. High-density surfaces were created ranging from 1,300 to 12,800 RUs to minimize nonspecific binding of ELP-GFP derivatives. Binding was measured with 60-s association phase and 600-s dissociation phase with either no regeneration, or surfaces were regenerated with two 30-s pulses of 50 mM NaOH. ELP-GFP derivatives were injected in duplicates from twofold dilution series with at least six different concentrations ranging from 0.28 to 60 M (depending on the polymer and its expected Kd); PBS was used as running buffer. Data were doubly referenced against the signal collected on the reference cell and responses generated on the active cells during buffer injections. Data were analyzed using Evaluation software and fit into a steady-state affinity binding model. Each reported affinity is an average from four to eight independent measurements.

The activity of recombinant trx and trx fusion proteins were determined using the Proteostat thioredoxin-1 activity assay (Enzo). Trx catalyzed reduction of insulin and consequent aggregation of insulin in the presence of dithiothreitol (DTT) was monitored by a fluorescent dye. Trx activity was determined using a standard curve using a concentration range of bacterial trx, and the activity of samples were determined to be within the linear range of the assay. Fluorescence emission was monitored using a Biotek spectrophotometric plate reader.

All experiments were performed in C57BL/6J mice in accordance with the guidelines of the Animal Care and Use Committee of Yale University. Recommendations from the Guide for the Care and Use of Laboratory Animals (49) were followed during these experiments.

The half-lives of ELP-GFP constructs were calculated from concentrations measured from blood samples collected over the course of a week. The experiments were initiated by injecting 120 L of 10 M ELP-GFP intravenously or subcutaneously. At indicated times, 2 L blood was collected from a tail puncture, and diluted 1:25 in heparin tubes. The blood sample was vortexed briefly and cells were pelleted by centrifugation (2 min at 14,000 g). The soluble fraction was collected and frozen at 20C until analysis. ELP-GFP concentrations of the samples were determined using a GFP ELISA Kit (Abcam, catalog #ab171581). The samples were diluted in PBS as needed, to ensure that the concentration fell within the quantifiable range of the standard curve.

To study the immunogenicity and biodistribution of ELP-GFP, 120 L of 10 M Alexa Fluor 647 labeled constructs were injected, and blood and organs were collected at indicated times. As positive control for an inflammatory response, 100 g LPS was injected, and an injection of PBS was performed as negative control. Organs were imaged using Amersham Imager 600 RGB, and signal visualization and quantification were performed with FIJI (https://imagej.net/software/fiji/). For cytokine quantification, blood was allowed to coagulate, and serum was collected. Cytokines were quantified from the serum samples using the BD CBA Mouse Inflammation Kit (Fisher Scientific, catalog #BD 552364).

Data supporting the findings of this work are available within the paper and its supporting information files. The strains and plasmid (sequences) have been deposited in GenBank or Addgene: Genetically recoded organism C321.deltaA (RRID: Addgene_48998), aminoacyl tRNA synthetase (RRID: Addgene_73545), ELP-GFP reporter (Genbank: KT996142). All other study data are included in the article and/or SI Appendix.

We thank Jesse Rinehart, Michael Grome, and members of the F.J.I. laboratory for discussions and feedback on the manuscript; Terence Wu (Yale West Campus Analytical Core) for technical support; and Dr. Mark Karpasas from the Ilse Katz Institute for Nanoscale Science & Technology at Ben-Gurion University for professional help with the intact mass spectrometry experiments. We acknowledge support from the Gruber Foundation (K.V.), NIH Grant K99EB019501 (to M.A.), the Dahlia Greidinger Anti Cancer Fund (M.A.), Ben-Gurion University (M.A.), NIH Grant R01GM117230 (to F.J.I.), NSF Grant MCB-1714860 (to F.J.I.), the Arnold and Mabel Beckman Foundation (F.J.I.), DuPont Inc. (F.J.I.), and Yale University (F.J.I.) for funding.

Author contributions: K.V., P.A.-G., D.H., W.M.S., M.A., and F.J.I. designed research; K.V., P.A.-G., M.K., D.H., A.G., F.Y., E.F.-S., and M.A. performed research; K.V., P.A.-G., D.H., E.F.-S., and M.A. contributed new reagents/analytic tools; K.V., P.A.-G., M.K., D.H., E.F.-S., W.M.S., M.A., and F.J.I. analyzed data; and K.V., W.M.S., M.A., and F.J.I. wrote the paper.

Competing interest statement: K.V., P.A.-G., M.A., and F.J.I. have filed patents describing this work. F.J.I. is a cofounder of Pearl Bio, Inc.

This article is a PNAS Direct Submission.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2103099119/-/DCSupplemental.

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Tuning protein half-life in mouse using sequence-defined biopolymers functionalized with lipids - pnas.org

The McKusick-Nathans Institute of Genetic Medicine and theDepartment of Genetic Medicineseekto further the understanding of human heredity and genetic medicine and use that knowledge to treat and prevent disease.

The Department of Genetic Medicineis working to consolidate all relevant teaching, patient care and research in human and medical genetics at Johns Hopkins to provide national and international leadership in genetic medicine. The Department of Genetic Medicineserves as a focal point for interactions between diverse investigators to promote the application of genetic discoveries to human disease and genetics education to the public. It builds upon past strengths and further develops expertise in the areas of genomics, developmental genetics and complex disease genetics. The Department of Genetic Medicineworks to catalyze the spread of human genetic perspectives to other related disciplines by collaboration with other departments within Johns Hopkins.

There are more than 300 dedicated employees in the Department of Genetic Medicine, fulfilling the Johns Hopkins tripartite mission of research, teaching and patient care. They include 45 full-time faculty, 15 residents, more than 70 graduate students and 200 staff.

All too often, when we see injustices, both great and small, we think, that's terrible, but we do nothing. We say nothing. We let other people fight their own battles. We remain silent because silence is easier. Qui tacet consentire videtur is Latin for 'Silence gives consent.' When we say nothing, when we do nothing, we are consenting to these trespasses against us.Roxane Gay

The indifferent and arrogant murder of George Floyd is but one of many searing examples of racism, oppression and sheer wickedness imposed on members of the African-American community over the last 400 years. Repeatedly, over these many years, periods of apparent progress have been undercut by horrific acts of racially-based evil that expose an underlying hard core of racial bias and systematic racism. The sadness, anger and frustration we all feel are compounded by the failure of our society to respond to these events with real and sustained justice. We cannot, however, let these events undermine our quest for meaningful and sustained progress towards correcting the systemic problems and beliefs leading to these events. To quote Martin Luther King Jr., Change does not roll in on the wheels of inevitability, but comes through continuous struggle.

How can we break out of this cycle of modest progress punctuated by horrific failures? The answers to this question are neither simple nor obvious. Success will require a sustained and multi-faceted effort from all of us. Some reactions seem obvious and personally attainable; we must treat all members of our society equally and fairly. In these difficult times, we much reach out to those directly affected with understanding, respect, and support. All of us must commit to and participate in these positive interactions. Beyond these responses of the moment, we must search for ways that we can change the social, economic and personal environment to minimize the likelihood of recurrence and maximize progress towards real equality for all. As geneticists, we treasure diversity and understand many of the biological factors underlying it. Perhaps, one special responsibility for us is to help others in society understand and value diversity and individuality.

As members of the Human Genetics program and Department of Genetic Medicine community, we recognize there are some among us who are more vulnerable to the biases illuminated by the death of George Floyd and many, many others; whose fear of an encounter with the police is amplified by personal and community experience; and whose experience of pain and suffering far exceeds what most of us can fully understand. To those most vulnerable in our Department of Genetic Medicinefamily, we stand with you and raise our voices to support you. We are ready to listen and act in pursuit of a learning environment of which you can be proud and a world into which you will move and feel free to change.

Finally, as we search for appropriate responses, we are grateful to have your voices, your guidance to help illuminate a path forward. We recognize and are encouraged by the outpouring of activism, passion, rage and love from our students, our department, our community and even our own families. We also recognize that this journey, which began centuries ago, will be long, sometimes uncomfortable and inelegant and studded with setbacks. We are, however, committed to do everything in our power to speed its progress.

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Department of Genetic Medicine | Johns Hopkins Medicine

By Tuyen Ong, MD, MBA, Ring Therapeutics and Flagship Pioneering

We spend so much time thinking about viruses that cause disease, especially over the last two years, but have you ever considered the fact that not all viruses are harmful? What if we could harness the power of relatively harmless viruses to solve major obstacles in medicine? For the first time ever, researchers are focusing on growing our understanding of a relatively understudied class of harmless viruses that live within all humans and could be the answer to many questions in genetic medicine.

Since its discovery, the human microbiome has served as a rich source of therapeutics and medical insights,1-3 but a lesser-known component of the microbiome, the virome, can potentially offer solutions to problems that face medicine today. The human commensal virome is composed of a plethora of viruses that live harmoniously within all of us.4 These commensal viruses often have broad tropisms, inhabiting a wide range of tissues, such as our eyes, hearts, and livers, all the while subverting the immune response that accompanies foreign viral infections.

Throughout history, researchers have discovered how to transform foreign viruses into life-saving therapies. This began as vaccines in which viral material prepares the immune system to fight a future infection and even led to using viruses to fight certain cancers. But one of the most recent advancements in using foreign viruses to fight human disease is gene therapy engineering viruses to replace or fix genetic defects. Though as game-changing as these viral-based gene therapies are, many barriers still limit their therapeutic potential.

Since the first U.S.-approved therapy for a genetic disease in 2017,5 researchers began ushering in an era of treatment possibilities for those with debilitating genetic diseases using foreign viral vectors.

Viral vectors use the capsid of a virus to encapsulate and deliver genetic cargo to the cell nucleus, and because viruses are so effective at entering host cells, they quickly became standard.6-7

The most common viral vectors in gene therapy today are adeno-associated viruses (AAVs), non-enveloped viruses belonging to the parvovirus family discovered in the 1960s.8-9 At the outset, AAVs were promising, offering a potential delivery vehicle that was tractable and relatively safe in preclinical models. Additionally, the viral biology of AAVs suggested prolonged safety profiles that would largely avoid mutagenesis, a likely result if the genetic material integrated within the host genome. Although some genomic integration has been observed in animal models,10-11 the AAV vector cargo largely remains as an episomal element within the nucleus, a positive in that theres less risk of insertional mutation but with a complementary downside whereby the cargo is diluted as cells grow and divide. This dilution requires redosing of the gene therapy in any tissue with dividing cells. However, once injected, the formation of neutralizing antibodies against the foreign AAV viral vectors has prevented safe redosing and resulted in the need for large, and often toxic, initial doses to be effective. These toxic doses have at times resulted in tragic outcomes in clinical trials.12-13 Additionally, natural prevalence of neutralizing anti-AAV antibodies in large proportions of the population (>40% in some serotypes14) precludes some patients from receiving even an initial dose of AAV-driven gene therapy.

Lentiviral vectors, a spherical enveloped RNA retrovirus, are another common viral vector in gene therapy today. Lentiviruses elicit a relatively weak immune response in animal models,15-16 and recent engineering successes have enhanced transduction of specific cell types.17 However, they are integrating viral vectors, which can provide long-term transgene expression, but also have a much higher risk of oncogenesis as a result of integration.18

Although viral vectors remain an ideal model for delivering genetic cargo because of their ability to efficiently enter host cells, the immunogenicity of AAVs and other vectors is a significant hurdle. The ideal viral vector platform would not elicit an immune response, would be potent, and would be diverse enough to infect targeted tissue types selectively.

The commensal virome may hold the solution. Recent research has shown how it may solve some of the greatest challenges remaining that are preventing the promise of gene therapy from reaching its full potential as a truly transformative treatment option.

Advances in metagenomic sequencing have revealed insights into the human virome with boundless value for gene therapy researchers. This includes exploring the diversity of a family of small commensal viruses called anelloviruses that have never been associated with disease.19-20

Almost every human maintains a diverse community of anelloviruses, called the anellome, that makes up the largest family of eukaryotic viruses within the human commensal virome.21

Anellovirus genomes do not integrate into the human genome, exhibit a broad yet specific tropism, and do not elicit a robust immune response, which allows them to persist in us for months or even years.19,22 These key facets make anelloviruses a promising new tool in our effort to overcome todays limitations in genetic medicine.

Anelloviruses are highly prevalent in humans, and many individuals often harbor a unique and dynamic anellovirus landscape.23 Additionally, a recent blood transfusion study utilizing our platform showed that anelloviruses are extremely diverse, highly transmissible, and persistent, with donor anelloviruses detected in recipients more than 200 days post transfusion (the last time point of data collection).23

Not only were anelloviruses shown to be both transmissible and persistent, but evidence also suggests that they are redosable. Despite significant genomic similarity between anelloviruses of recipients and donors, the anelloviruses from both donor and recipient co-exist within the recipient without causing any neutralizing immune response for an extended period of time.23 This critical facet of anellovirus biology could finally permit redosing of a viral vector in gene therapy.

Building on this data, Ring scientists set out to harness the unique anellovirus biology for therapeutic application. For a newly identified anellovirus genome, the team can recreate it in vitro as a fully synthesized recombinant human anellovirus, then recover it using traditional viral purification methods, marking the first time human anelloviruses have been produced in vitro.24

These recombinant anelloviruses can also be vectorized, loading the anellovirus capsid with chosen DNA cargo that can be successfully transduced into human cells in vitro.24 Ring dubs this novel viral vector an Anellovector.

Although natural anelloviruses do not elicit a robust immune response, the question remained whether humans maintain immunity against synthesized Anellovectors. Similar to natural anelloviruses and to no surprise to the Ring team, preclinical studies of its initial Anellovectors identified low preexisting immunity against it in a pool of thousands of human donor sera,24 validating the potential utility of Anellovectors as the next generation of gene therapy vectors.

Anellovectors harness the unique biology of human anelloviruses and have strong therapeutic potential because of five key features:

Anelloviruses are ubiquitous within the human body and, having co-evolved with humans for millennia, largely avoid triggering any significant immune reactions. Transfusion of similar anelloviruses between human patients showed persistence without a deleterious immune response.23 Through harnessing the unique feature of immune stealth of anelloviruses, Anellovectors have the potential to treat patients requiring redosing, such as those suffering from spinal muscular atrophy and hemophilia.

Greater than 40% of patients maintain a natural prevalence of some serotypes of anti-AAV neutralizing antibodies,14 precluding them from receiving even an initial dose of any AAV-driven gene therapy. Because humans maintain low preexisting immunity to anelloviruses and Anellovectors, their use may offer the opportunity for these patients to receive a potentially life-saving gene therapy treatment.

Many tissue types are elusive to current gene therapy vectors and are deemed unreachable. However, anelloviruses exhibit a broad tissue tropism, being found from head to toe of most humans,19 including those that are currently unreachable. Therefore, Anellovectors can be designed through harnessing specific anelloviruses that exhibit a targeted tropism. This specificity opens the door for potential solutions to a broad range of genetic diseases across tissue types, including the liver, offering a safer option with no hepatotoxicity.

Current virus-based gene therapy platforms, including some instances with AAVs, can randomly integrate within the human genome, disrupting the function of important genes and sometimes even causing oncogenesis. As with anelloviruses, Anellovector cargos remain as episomes in the nucleus and do not affect the host genome, minimizing the potential risk and making for a safer form of gene therapy.

Because Anellovectors can be synthesized and vectorized, Anellovectors can be engineered to accommodate a wide variety of therapeutic modalities, from single-stranded DNA for gene therapies to potentially enabling delivery of larger DNA products through packaging of mRNA.

Through a better understanding of the commensal virome, theres hope for a brighter future for gene therapy. This promising new technology may help gene therapy reach its full potential by overcoming the current limitations with repeat dosing, toxicity, and limited tropism.

Rings research team has uncovered and characterized the worlds largest collection of commensal anelloviruses and is harnessing those best suited for specifically targeting a wide array of diseases with significant unmet needs. The unique biology of anelloviruses may indeed provide a foundation for an unparalleled new gene therapy platform and the resulting new class of genetic medicines.

References

About The Author:

Tuyen Ong, MBA, MD, is CEO of Ring Therapeutics and CEO/Partner of Flagship Pioneering. He is a physician and bioentrepreneur. Prior to joining Ring Therapeutics in 2020, he served as senior vice president at Biogen and as Chief Development Officer at Nightstar Therapeutics until its acquisition by Biogen. During this time, he was involved with the companys public listing on the NASDAQ, corporate and gene therapy strategy, investor, and M&A activities. Ong brings more than 20 years of clinical and drug development experience from both large pharma and biotech, working in the fields of genetics, ophthalmology, and rare disease at PTC Therapeutics Inc., Bausch and Lomb Inc., and Pfizer. He is a member of the Royal College of Ophthalmologists and a Churchill Fellow.

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Harnessing The Human Virome For The Next Generation Of Gene Therapy Vectors - Clinical Leader

New research finds a strong association between a rare genetic mutation and early-onset heart failure and hospitalization among Black Americans.Researchers found Black patients who develop heart failure and carry this genetic mutation develop the condition four years earlier than those not carrying it.

Dr. Ambarish Pandey, co-author of the study and assistant professor of internal medicine at the University of Texas Southwestern Medical Center, said the study raises the question: Should Black Americans be screened for this genetic mutation?

That's a question that I cant answer in just one study, he said. But I think it raises this broader question of: Is there benefit in screening for this abnormal mutation in Black patients, particularly in those who may be identified early on and can benefit from more aggressive risk modification for things like blood pressure control and other factors that contribute to risk of heart failure?

The genetic mutation known as TTR V142I allows protein to build up inside the heart, which can make it harder for the heart to contract and relax. That eventually can cause part of the organ to die a condition known as transthyretin amyloid cardiomyopathy.

Several prior studies linked the genetic mutation to this condition, but those studies did not explore the association with adverse clinical outcomes, including heart failure and hospitalization rates, Pandey said.

Researchers followed thousands of study participants for 12 years

This new study is the first to look at an exclusively Black cohort, he said. Thats significant because the genetic mutation is much more prevalent in people of African descent.

Pandey, along with researchers at several U.S. medical schools, followed a cohort of nearly 3,000 people of African descent over a period of 12 years and identified more than 200 patients who developed heart failure. The researchers found people with the mutation who experienced heart failure were more likely to have negative health outcomes, including early-onset heart failure, heart muscle injury and hospitalization.

Heart failure occurs when the heart cant pump enough blood to the bodys vital organs. Without close monitoring and management, the condition can be lethal. More than 6 million Americans are living with heart failure, according to the U.S. Centers for Disease Control and Prevention.

Health issues like obesity, high blood pressure and diabetes can increase a persons risk of heart failure. African Americans have higher rates of heart failure, hospitalization and death from the disease, according to national data.

This study was done on Black Americans participating in the Jackson Heart Study the longest and largest study looking at cardiovascular disease in Black Americans. The researchers followed the participants from 2005 to 2016. The process included heart imaging and blood tests.

Among the other significant findings: Even when heart imaging didnt turn up evidence of injury to the organ in study participants with the genetic mutation, blood tests that look for troponin, a marker of tissue damage, did. Those with the genetic mutation had significantly higher amounts of this protein in their blood.

The study opens a new avenue of investigations to further develop drugs and treatments, even prevention for patients [who] have this genetic predisposition to disease, said Dr. Patrice Desvigne-Nickens, program director at the National Heart, Lung and Blood Institute, which funded the research.

Desvigne-Nickens, who was not part of the study, said there are FDA-approved treatments that prevent heart failure when the heart begins to show signs of damage. But it remains unknown whether someone who carries the genetic mutation, but does not yet have abnormal amyloid protein deposits, could reduce their risk of heart failure by receiving those treatments.

Still, Desvigne-Nickens said these findings suggest that patients who already have heart disease may benefit from genetic testing.

If a Black patient is found to have this mutation, their physician and providers need to carefully monitor for signs and symptoms of heart failure, she said. Additionally, because this is a genetic marker, family members of patients with this mutation may also benefit from getting tested.

More research and improved health care access needed

Pandey said further research is needed before genetic testing is recommended for all people of African descent. He said in order to improve health outcomes, increasing access to genetic testing isnt the most pressing issue.

Access to care is a big issue in Black communities, Pandey said. So, forget about implementing genetic testing for amyloidosis. I think if I had to pick what to improve access to, I would improve access to blood pressure control, diabetes control, and things that are more prevalent and more common, and also associated with the risk of heart failure just as much.

Researchers dont know why this genetic mutation exists almost exclusively in patients of African descent. The vast majority of peoples genetic composition is consistent across races. But some genetic mutations show up with greater frequency among certain populations.

Population genetics and origins are important, because there is a selection having to do with your environment that determines the frequency of certain genes and variations, Desvigne-Nickens said.

For instance, the sickle cell trait a genetic trait that can cause sickle cell anemia, a blood condition that primarily affects people of African descent seems to be related to the prevalence of malaria in Africa.

Still, Desvigne-Nickens said the role genetic variations play in peoples health is far less significant than environmental factors. Long-standing socioeconomic and disparities in health care access facing Black Americans are among the contributing factors to many health conditions, she said.

The majority of the largest contributors to how long you live, and how healthy your life is, have to do with your behavior and the environment in which you live, she said. So, genetic testing doesn't absolve you of responsibility for healthy habits, healthy living and making sure that the environment is not hostile.

Desvigne-Nickens said this study is one type of precision medicine, which considers a persons genetics, environment and lifestyle to inform potential treatments. The approach could help address long-standing health disparities and the participation of African American patients makes this possible.

A real shout out to them, the participants. These are African Americans [who] participate in these studies and allow themselves to be tested because that information then can be used to help them, she said. Research is not something that has been welcome in communities of color, because research in the past has represented some level of exploitation and breach of trust for a variety of reasons.

As a physician of color herself, Desvigne-Nickens said better representation in medical and research spheres would help cultivate more trust and participation of communities of color in crucial research.

This story comes from a reporting collaboration that includes the Indianapolis Recorder and Side Effects Public Media a public health news initiative based at WFYI. Follow Farah on Twitter: @Farah_Yousrym.

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While some people seem to just stay young longer, others age prematurely. Your chronological age of course cant be changed, but research suggests the biological processes that drive aging may in fact be malleable.

Understanding those processes is the goal of the new Potocsnak Longevity Institute at Northwestern Universitys Feinberg School of Medicine.

Dr. Douglas Vaughan, director of the new institute and chair of medicine at Northwestern, discusses aging, the institute and its goals.

Below, a Q&A with Vaughan.

Explain the concept of physiological age versus chronological age. How do you measure physiological age?

Thats a very good question. And its very pertinent to the conversation that were having and the idea that were presenting. It turns out there are a variety of physiological measures that we can perform on any human being that actually change with age. And its the integrated and cumulative measurement of a variety of different parameters that allows us to make a calculation of someones physiological or biological age as opposed to their chronological age.

There are also very specific molecular markers that change with age.

Patients at the new institute will undergo a battery of tests to determine their physiological age. How does that testing differ from what people typically get for an annual physical?

Thatsa really important question. We will actually focus on measures that change with age. So for example we will measure grip strength, we will measure hearing, we will measure heart rate variability, we will measure the capacity of your blood vessels to dilate, we willmeasure how far you walk in five minutes. All these kinds of things arent part of a routine physical exam. Theyre sort of on the edges of it, but theyre not specifically quantified and calculated.

The ultimate aim of your research is to allow people to live well longer. What do you think is feasible when you start looking at how much longer we might be able to extend human lifespans? Are we talking years, decades? Obviously, weve already made significant gains over the last century. How much more do you think we might be able to achieve?

Well, we were making gains until the pandemic hit. I think the average lifespan in the United States has dropped by two years since the pandemic hit. I think we can extend the health span of people, maybe another 10-15 years. I dont think our goal is to have people to make it to 120 or 150. But if we can push back age-related illnesses, whether its cancer or cardiovascular or neurodegenerative disease or lung disease and have people live a fuller, more productive, healthier life, thats the goal of all this. And I think thats within reach.

Youve done research on a community of Amish in Indiana who have a unique genetic variant that seems to allow them to live about 10 years longer than those who dont have the variant. When you look at the science of aging, how much is it about the genetic lottery that we all are dealt? How much is changeable, and how much is not?

Well thats an extremely complicated question. This is conjecture on my part. We certainly dont change the primary components of our genetic makeup, but our genes do change over time.

Our DNA gets modulated or chemically changed and it changes the function of our DNA. One of the processes there that contributes to that is called methylation. And specific patterns of methylation of our DNA predict the development of aging-related illnesses and lifespan. So I think that we actually have the capacity to impact upon that process as well as lifestyle interventions or other therapeutic interventions that could impact upon the aging that we all experience.

In terms of like the specific genetic trait identified in these Amish folks in Indiana, is that something that could be used and applied to people without that genetic variation to help them live longer as well?

So the genes, the genetic variants that this Amish population carries codes for a protein that circulates in our blood. And the carriers of that genetic variant have half the normal level of the protein. And its very easy to measure.

I can measure it in anybody. There are already drugs in clinical trials that lower the level of the protein in human beings. So you can simply take a drug by mouth and lower your level of that specific protein. And there will be other examples that are discovered like that in the next few years. This community is very unique in that it harbors this remarkable genetic variant. Theres no other population quite like it in the world. . Were still investigating the broad impact of that genetic variant. It seems to touch on almost every system in the body.

Theres also a connection between HIV research and aging research. Explain that linkage.

Thats a really important component of our story in regards to the generous individuals that provided the money to create the institute, John Potocsnak and his family, who are very interested in HIV.

HIV was a lethal disease in the 80s. Now its a chronic disease, but were learning that many people with chronic HIV infections look like they age more rapidly than individuals that dont have HIV. I think that biology is really fascinating. It probably provides some insight into the aging process across all human beings, and hopefully well be able to develop some intellectual reciprocity by studying HIV and aging, and aging in human beings that dont have that infection.

What is your ultimate goal for the institute? Where do you hope the research will be in say 10 years?

We really want to contribute to the process of understanding the biology of aging and how it impacts upon the human condition. We hope that we will make a meaningful contribution to extending the healthy lifespan of our species and its as simple and as powerful as that.

And do you think that the benefits of that research and whatever therapies or treatments come out of it are likely to be widely available? Do you think these are the kind of things that could someday be covered on health insurance?

Id be very disappointed if it wasnt. I mean there are there are already drugs out in the marketplace that are suspected to potentially have anti-aging properties. One of the drugs that is commonly mentioned is Metformin thats broadly administered to people with diabetes. But it may have other kinds of interesting properties and its extremely cheap.

So, if a drug like that could be actually beneficial in delaying aging related morbidity that would be broadly applicable to the population. The drug that I talked about thats being developed to block a protein, its not going to necessarily be very expensive. Its not going to be thousands of dollars a month to take a drug like that. Itll be dollars a month.

Interview has been condensed and edited.

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Northwestern's New Longevity Institute Aims to Decode the Mysteries of Aging - WTTW News

Its that time of year again! With 2021 behind us, were going down memory lane to highlight biotech innovations that shaped the yearwith impact that will likely reverberate for many years to come. Covid-19 dominated the news, but science didnt stand still.

Take gene editing. CRISPR spun off variations with breathtaking speed, expanding into a hefty toolbox packed with powerhouse gene editors far more efficient, reliable, and safer than their predecessors. CRISPRoff, for example, hijacks epigenetic processes to reversibly turn genes on and offall without actually snipping or damaging the gene itself. Prime editing, the nip-tuck of DNA editing that only snipsrather than fully cuttingDNA received an upgrade to precisely edit up to 10,000 DNA letters in a variety of cells. Twin prime editing can rework entire genes. These powered-up CRISPR tools now make it possible to tackle previously untouchable genetic disorders.

Yet were still only scratching the surface of gene editing. Peeking into the CRISPR family tree, scientists found a vast universe of alternative CRISPR-like systems to further explore. AI is now helping identify new CRISPR proteinsand their kill switch. Other ideas jumped ship from CRISPR altogether, tapping into another powerful bacterial system to edit millions of DNA sequences without breaking a single DNA strand. Without doubt, the gene editing toolbox will keep expanding.

In other news, quantum mechanics hooked up with neuroscience to speed up AI. AI is now designing its own hardware chips at Google in an efficient full circle. Hopping into our own brains, in a stunning proof-of-concept, AI-powered brain implants were able to fight depression, with ongoing work to treat chronic pain and translate the brains electrical signals from thought to text. In the medical world, a fierce debate on an Alzheimers treatment sparked a new round of alluring ideas to tackle and tame our long-time mind-eating foe.

Theres a ton more. But here are the top three advances thatll keep reshaping biotech far past 2021, with some runners-up.

I know, I know. Were all tired of hearing about Covid-19 and vaccines. Yet their remarkable ability to fight a completely novel infectious virus is nothing short of miraculous. It also showcased the power of the decades-old technology that previously languished in labs, with a platform thats far faster, simpler, and more adaptable than any previous vaccine technology. Because they no longer rely on physical target proteins from a virusrather, just the genetic code for those proteinsdesigning a vaccine just requires a laptop and some ingenuity. The era of the digital vaccine is here, wrote a team from GlaxoSmithKline.

To enthusiasts, mRNA vaccines could transform current treatments for a wealth of diseases, and the field is exploding. Moderna, for example, launched an HIV vaccine humantrial in August to begin assessing its safety, tackling a virus thats escaped classic vaccine tactics for four decades. Along with the National Institutes of Health (NIH), the company also published data on an HIV vaccine candidate that lowered the chance of infection by nearly 80 percent in monkeys, with all subjects developing antibodies against 12 tested strains of HIV. Its no small featthe HIV target, Env, is a formidable target due to its complexity and is coated with a sugar armor to mask vaccine target points. The mRNA vaccine offers new hope.

Viruses aside, mRNA vaccines also represent a new solution to autoimmune or neurodegenerative diseases. BioNTech, the partner of Pfizer for developing Covid-19 vaccines, is applying the technology to tackle multiple sclerosis (MS). In MS, the immune system gradually strips away the insulation on nerve fibers, causing gradual and irreversible damage. Initial results in mice are positive, with the approach highly flexible, fast, and cost efficient, while potentially being personalized to each patient.

Further down the pipeline are mRNA vaccines that tackle cancer or those that deal with antibiotic resistance. Whether the tech can solve some of our toughest diseases remains to be seen, but the field is on a roll.

CRISPRs long been touted as a tool that can radically transform gene therapy. Earlier studies used the gene editing tool to bolster immune T-cells, transforming them into super soldiers that enhance their fight against blood cancers (CAR-T therapy). The tool also scored successes in battling anemia and other symptoms in patients with blood disorders. The down side was that cells needed to be gene-edited outside the body and infused back into the bloodstream. This year elevated CRISPR to the ultimate goal: directly editing genes inside the body, opening the door to curing hundreds of disorders resulting from faulty genetic code.

In a breakthrough, one trial from University College London edited a mutated gene in the liver that eventually leads to heart and nerve damage. Unlike previous attempts, here the CRISPR machinery was delivered into the bloodstream with a single infusion to switch the gene off, sharply decreasing the production of the mutant protein in six patients. Another trial snipped a dysfunctional gene that causes blindness. By directly injecting the treatment into the retina, volunteers were able to better sense light.

Both are edge cases. For the liver trial, CRISPR was delivered using lipid nanoparticleslittle fatty space shipsthat have an affinity for the liver, with more transient gene-editing effects. And unlike the retina, most of our bodys tissues arent immediately accessible to a simple injection. But as proofs of concept, the trials finally bring CRISPR into a vast world of gene-editing possibilities inside the body. Along with advances in delivery, CRISPRand its many upgradesis set to treat the untreatable.

The first few hours and days of a human embryos development are a black boxone we need to crack. Understanding early pregnancy is key to limiting birth defects and pregnancy loss, and improving assistive reproduction technologies.

The problem? Early embryos are hard to come by, and carry significant ethical and legal challenges. This year, several studies circumvented these problems, instead transforming skin cells into blastocysts, a cellular structure that resembles the very first stage of a human embryo.

Torpedoing the usual sperm meets egg narrative, the studies engineered the first complete model of the human embryo using embryonic stem cells and skin cellsno reproductive cells needed. Bathed in a nutritious liquid, the cells developed into blastocysts, containing cell types that eventually lead to all lineages to build our bodies. The artificial embryos are genetically similar to natural ones, stirring up debate on how long they should be allowed to develop. The nightmare scenario? Imagine a mini-brain growing inside an embryo made out of skin cells!

For now thats technically impossible, but the ethical quandary has stirred up concern at the International Society for Stem Cell Research (ISSCR), which governs research related to human stem cells and embryos. Yet surprisingly, this year, they relaxed the 14-day rule for culturing embryos, giving permission to push embryo research past two weeks. With relaxed guidelines, upcoming studies could reveal what happens to a human embryo after implanting into the uterus, and gastrulationwhen genetic cues lay out the bodys overall patterning and set the stage for organ development.

Its a decision mired in controversy, but provides an unprecedented opportunity to revise IVF and, for the first time, examine the first stages of human development. Its also bound to raise ethical quandaries: what if the embryosnatural or artificialbegin developing neurons that fire, or heart cells that pulse? As artificial blastocysts increasingly embody their biological counterparts, one thing is clear: with great power comes great responsibility.

AI predicting proteins: DeepMind and the University of Washington both engineered AI that can solve the structure of a protein based purely on its genetic code. Its a once in a generational advance, a breakthrough of the year, and a tool thatll change structural biology forever. Updates to the original AI can now also predict protein complexesthat is, how one protein unit interacts with anotherand even their function. AI is also beginning to solve RNA structure, the messenger that bridges DNA to proteins. From synthetic biology to drug development, the impact is yet to come.

AI-designed drugs: Its been a long time in the making, but the hype is now real. This year, Alphabet, Googles parent company, launched a new venture called Isomorphic Labs to tackle a new world of drug development using AI. Powerful algorithms are making it increasingly easy to screen drug candidates from millions of chemicals. And the first AI-discovered drug is now going into clinical trials in a safety test for a lung disease that irreversibly degrades the organs function. Its a significant milestone, and the trial may pave the road for the first AI-discovered, human-tested drug that treats diseases.

In another year of living with Covid-19, its clear that the pandemic cant hold science down. I cant wait to share the good, the weird, and (holds breath) more breakthroughs of a generation biotech stories in 2022.

Image Credit: Schferle / 94 images / Pixabay

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These 2021 Biotech Breakthroughs Will Shape the Future of Health and Medicine - Singularity Hub

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If the pandemic had happened ten years ago, what would it have looked like? Doubtless there would have been many differences, but probably the most striking would have been the relative lack of genomic sequencing. This is where the entire genetic code or genome of the coronavirus in a testing sample is quickly read and analysed.

At the beginning of the pandemic, sequencing informed researchers that they were dealing with a virus that hadnt been seen before. The quick deciphering of the viruss genetic code also allowed for vaccines to be developed straight away, and partly explains why they were available in record time.

Since then, scientists have repeatedly sequenced the virus as it circulates. This allows them to monitor changes and detect variants as they emerge.

Sequencing itself is not new whats different today is the amount taking place. Genomes of variants are being tested around the world at an unprecedented rate, making COVID-19 one of the most highly tested outbreaks ever.

With this information we can then track how specific forms of the virus are spreading locally, nationally and internationally. It makes COVID-19 the first outbreak to be tracked in near real-time on a global scale.

This helps with controlling the virus. For example, together with PCR testing, sequencing helped reveal the emergence of the alpha variant in winter 2020. It also showed that alpha was rapidly becoming more prevalent and confirmed why, revealing that it had significant mutations associated with increased transmission. This helped inform decisions to tighten restrictions.

Sequencing has done the same for omicron, identifying its concerning mutations and confirming how quickly its spreading. This underlined the need for the UK to turbocharge its booster programme.

The road to mass sequencing

The importance of genomic sequencing is undeniable. But how does it work and how has it become so common?

Well, just like people, each copy of the coronavirus has its own genome, which is around 30,000 characters long. As the virus reproduces, its genome can mutate slightly due to errors made when copying it. Over time these mutations add up, and they distinguish one variant of the virus from another. The genome of a variant of concern could contain anywhere from five to 30 mutations.

The viruss genome is made from RNA, and each of its 30,000 characters is one of four building blocks, represented by the letters A, G, C and U. Sequencing is the process of identifying their unique order. Various technologies can be used for this, but a particularly important one in getting us to where we are is nanopore sequencing. Ten years ago this technology wasnt available as it is today. Heres how it works.

First the RNA is converted to DNA. Then, like a long thread of cotton being pulled through a pinhole in a sheet of fabric, the DNA is pulled through a pore in a membrane. This nanopore is a million times smaller than a pin head. As each building block of DNA passes through the nanopore, it gives off a unique signal. A sensor detects the signal changes, and a computer program decrypts this to reveal the sequence.

Amazingly, the flagship machine for doing nanopore sequencing the MinION, released by Oxford Nanopore Technologies (ONT) in 2014 is only the size of a stapler; other sequencing techniques (such as those developed by Illumina and Pacific BioSciences) generally require bulky equipment and a well-stocked lab. The MinION is therefore incredibly portable, allowing for sequencing to happen on the ground during a disease outbreak.

This first happened during the 2013-16 Ebola outbreak and then during the Zika epidemic of 2015-16. Pop-up labs were set up in areas lacking scientific infrastructure, enabling scientists to identify where each outbreak originated.

This experience laid the foundation for sequencing the coronavirus today. The methods honed during this time, in particular by a genomics research group called the Artic Network, have proved invaluable.

They were quickly adapted for COVID-19 to become the basis on which millions of coronavirus genomes have been sequenced across the globe since 2020. Nanopore sequencing of Zika and Ebola gave us the methods to do sequencing at a never-before-seen scale today.

That said, without the much larger capacity of the benchtop machines from Illumina, Pacific Biosciences and ONT, we wouldnt be able to capitalise on the knowledge gained through nanopore sequencing. Only with these other technologies is it possible to do sequencing at the current volume.

What next for sequencing?

With COVID-19, researchers were able to monitor the outbreak only once it had started. But the creation of rapid testing and screening programmes for other new diseases, as well as the infrastructure to conduct widespread sequencing, has now begun. These will provide an early warning system to prevent the next pandemic taking us by surprise.

For instance, in the future, surveillance programmes may be put in place to monitor wastewater to identify disease-causing microbes (known as pathogens) present in the population. Sequencing will allow researchers to identify new pathogens, allowing an early start on understanding and tracking the next outbreak before it gets out of hand.

Genome sequencing also has a role to play in the future of healthcare and medicine. It has the potential to diagnose rare genetic disorders, inform personalised medicine, and monitor the ever-increasing threat of drug resistance.

Five to ten years ago, scientists were only just beginning to trial sequencing technology on smaller viral outbreaks. The effects of the past two years have resulted in a huge increase in the use of sequencing to track the spread of disease. This was made possible by technology, skills and infrastructure that have developed over time.

COVID-19 has caused untold damage worldwide and affected the lives of millions, and were yet to see its full impact. But recent advances particularly in the field of sequencing have no doubt improved the situation beyond where wed otherwise be.

Angela Beckett, Specialist Research Technician, Centre for Enzyme Innovation, and PhD Candidate in Genomics and Bioinformatics, University of Portsmouth and Samuel Robson, Reader in Genomics and Bioinformatics, and Bioinformatics Lead, Centre for Enzyme Innovation, University of Portsmouth

This article is republished from The Conversation under a Creative Commons license. Read the original article.

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How COVID-19 transformed genomics and changed the handling of disease outbreaks forever - Down To Earth Magazine

LEXINGTON, Mass., Jan. 4, 2022 /PRNewswire/ -- LogicBio Therapeutics, Inc. (Nasdaq:LOGC), a clinical-stage genetic medicine company, today announced that president and chief executive officer, Fred Chereau, will participate in a fireside chat at the virtual H.C. Wainwright Bioconnect Conference being held January 10-13, 2022. The pre-recorded presentation will be available for on-demand viewing beginning at 7:00 a.m. ET on Monday, January 10, 2022.

A webcast of the presentation will be made available on the Investors section of the Company's website at http://www.logicbio.com/investors. The webcast replay will be available for approximately 30 days.

About LogicBio Therapeutics

LogicBio Therapeutics is a clinical-stage genetic medicine company pioneering genome editing and gene delivery platforms to address rare and serious diseases from infancy through adulthood. The company's genome editing platform, GeneRide, is a new approach to precise gene insertion harnessing a cell's natural DNA repair process potentially leading to durable therapeutic protein expression levels. The company's gene delivery platform, sAAVy, is an adeno-associated virus (AAV) capsid engineering platform designed to optimize gene delivery for treatments in a broad range of indications and tissues. The company is based in Lexington, MA. For more information, visit http://www.logicbio.com, which does not form a part of this release.

Investor Contacts: Stephen Jasper Gilmartin Group 858-525-2047 stephen@gilmartinir.com

Media Contacts: Adam Daley Berry & Company Public Relations W:212-253-8881 C: 614-580-2048 adaley@berrypr.com

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SOURCE LogicBio Therapeutics, Inc.

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