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Background: Several first-generation gene therapies are currently in clinical trials for Haemophilia A. These trials have to date exhibited varying results including issues with large patient to patient variation in FVIII levels and lack of durability. It is hypothesized that these challenges may, in part, be caused by the large size of the F8 transgenes used, resulting in genome sizes surpassing the optimal packaging capacity of AAV (~4.7kbp). We hypothesised that the oversize AAV vectors contain truncated genomes which cause batch to batch variations and decrease the quality of the resulting drug product. Aim: Armed with clinical success with our proprietary AAVS3 capsid in Haemophilia B (Nathwani et al, 2018), we designed novel, next generation potent AAV-F8 vectors with genomes of less than 4.9kbp to increase the quality of the resulting product and the predictability of FVIII expression. Methods: We deployed several methods to achieve our goal, including optimisations to the 'F8-SQ' coding sequence and optimisations of components of the expression cassette from the perspective of balancing size and potency of the resulting vectors. Over 20 optimized gene sequences, 10 different signal peptides, more than 60 short liver-specific promoters with a size of less than 270bp, and several shorter FVIII coding sequences were designed. We evaluated the resulting constructs for potency in human liver cell line Huh7 cells as well as in C57BI/6 mice. In addition, a range of different deletion lengths encompassing the B-domain were assessed for their in vitro and in vivo efficiency. Selected potent constructs were further evaluated in haemophilia A mice, and their haemostatic ability was demonstrated in the classical tail clip model. Results: All elements of the expression cassette were optimized to generate a construct with more than 70-fold higher expression than the wild-type FVIII construct as evaluated in the human liver cell line Huh7. Importantly, we were able to develop a series of short promoters ( Conclusion: We have developed several highly potent lead candidates with sizes of less than 4.9kbp, some being less than 4.75kbp in length. These candidates all have vector genomes markedly shorter in length than AAV-FVIII candidates currently in development and amongst these, we have selected the top best candidate, FLT210, for further development. Disclosures Kia: Freeline Therapeutics: Employment, Equity Ownership. Comper:Freeline: Employment, Equity Ownership. Correia:Freeline: Employment, Equity Ownership. Casari:Freeline: Research Funding. Negash:Freeline: Employment, Equity Ownership. Portillo:Freeline: Employment, Equity Ownership. Pandya:Freeline: Employment, Equity Ownership. Dodev:Freeline: Employment, Equity Ownership. Schulze:Freeline: Employment, Equity Ownership. Sonntag:Freeline: Employment, Equity Ownership. Kober:Freeline: Employment, Equity Ownership. Cocita:Freeline Therapeutics: Employment, Equity Ownership. Chisari:Freeline Therapeutics: Employment, Equity Ownership. Alade:Freeline: Employment, Equity Ownership. Shehu:Freeline: Employment, Equity Ownership. Jeyakumar:Freeline Therapeutics: Employment, Equity Ownership. Khinder:Freeline: Employment, Equity Ownership. Foley:Freeline: Employment, Equity Ownership. Dane:Freeline: Employment, Equity Ownership. McIntosh:Freeline Therapeutics: Consultancy, Equity Ownership. Ollerton:Freeline: Employment. Christophe:Freeline: Research Funding. Denis:Freeline: Research Funding. Corbau:Freeline: Employment, Equity Ownership.

Introduction: Gaucher disease (GD), one of the most common lysosomal storage disorders, is an autosomal recessive condition resulting from mutations in the GBA gene that codes for the b-glucocerebrosidase (GCase) enzyme. Over 90% of patients have type 1 GD, which is characterised by lipid engorged macrophages (known as Gaucher cells) in multiple organs, including spleen, liver and bone marrow, with no overt involvement of the central nervous system (CNS). The current standard of care for type 1 GD patients includes enzyme replacement therapy (ERT), which provides good overall therapeutic benefit. However, ERT is administered intravenously every other week, resulting in a high cumulative cost and a significant treatment burden. Furthermore, disease manifestations, such as pulmonary and skeletal disease, remain unresolved with ERT. Gene therapy is emerging as a very promising avenue of treatment for various monogenic disorders and has the potential to provide sustained levels of GCase enzyme expression after a single treatment. Here we have evaluated liver-directed gene therapy in vitro and in vivo for the treatment of GD. Methods: Adeno-associated virus (AAV) constructs were optimised to express full-length wild-type GCase protein (GBA AAV) and packaged in AAV8 capsids for in vivo mouse studies, or our novel AAVS3 capsid for in vitro studies in a human cell line. GCase activity was determined fluorometrically with 4-Methylumbelliferyl-β-D-glucopyranoside and activity was based on a 4-methylumbelliferone standard curve. Levels of GCase in plasma, and uptake in GD target organs were compared between our GBA AAV optimized construct and ERT treatment with velaglucerase alfa (VPRIV®) in C57BL/6 wild type mice. Doses used ranged from 2x109 to 2x1012 vg/kg for GBA AAV constructs and 60 U/kg for ERT. Results: Our initial proof of concept studies for liver-directed AAV gene therapy of GD used an AAV construct encoding the native full-length human GBA cDNA (RC-04-01). After a single intravenous injection into mice, RC-04-01 led to a dose-dependent expression of GCase in liver and robust levels of enzymatically active GCase in plasma. Based on these preliminary data, 37 GBA AAV constructs with optimisations to the coding sequence and changes to the promoter, signal peptide, and polyA sequences were designed and evaluated in vitro for GCase production. Among them, 6 constructs outperformed RC-04-01 and were further tested in mice. Construct RC-04-26 showed the highest GCase activity levels both in vivo and in vitro. At a dose of 2x1012 vg/kg, RC-04-26 showed plasma active GCase levels up to 9.4-fold higher than RC-04-01. Upon GBA AAV infusion, plasma GCase levels were steady and sustained for the duration of the entire study period (9 months). In addition, RC-04-26 infusion into mice resulted in robust uptake of GCase by cells in spleen, bone marrow and lung, demonstrating that liver-produced GCase is taken up by macrophages present in the GD target organs. Lastly, GCase bioavailability was evaluated after a single administration of RC-04-26 or ERT. With velaglucerase alfa, GCase was rapidly cleared from the bloodstream and tissues. However, RC-04-26 resulted in sustained and steady levels of GCase, with an overall bioavailability of GCase uptake over 170-fold higher than with ERT. Conclusions: Our current data support the hypothesis that a single administration of an optimised liver directed GBA AAV vector results in sustained elevation of GCase in the bloodstream and higher level of GCase bioavailability for uptake into macrophages than velaglucerase alfa. This observation supports further development of AAV gene therapy for Gaucher disease with the potential for enhanced therapeutic benefit from a one-off administration. Disclosures Miranda: Freeline Therapeutics: Employment, Equity Ownership. Canavese:Freeline Therapeutics: Employment, Equity Ownership. Chisari:Freeline Therapeutics: Employment, Equity Ownership. Pandya:Freeline: Employment, Equity Ownership. Cocita:Freeline Therapeutics: Employment, Equity Ownership. Portillo:Freeline: Employment, Equity Ownership. McIntosh:Freeline Therapeutics: Consultancy, Equity Ownership. Kia:Freeline Therapeutics: Employment, Equity Ownership. Foley:Freeline: Employment, Equity Ownership. Dane:Freeline: Employment, Equity Ownership. Jeyakumar:Freeline Therapeutics: Employment, Equity Ownership. Sheridan:Freeline Therapeutics: Employment, Equity Ownership. Corbau:Freeline: Employment, Equity Ownership. Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Introduction: AAV-mediated gene transfer of blood coagulation Factor IX (FIX) has been established as a safe and long-term treatment for patients suffering from severe hereditary Haemophilia B. A gain-of-function F9 transgene (F9-R338L; Padua) has recently been used to achieve higher functional levels of FIX, effectively eliminating the need for regular prophylaxis. The naturally-occurring R338L Padua mutation is situated in the catalytic domain of FIX on a helical side loop (region 332-339) that is involved in FVIIIa-mediated stimulation of substrate turnover. Here, we examined if a single amino acid substitution of a lysine at position 301 leads to gain of function. This basic residue sits adjacent to the 332-339 loop on an exposed helical segment (292-303) that has been implicated to interact with the FVIIIa A2 domain in the FIXa-FVIIIa tenase complex. Methods: We examined the lysine at position 301 (numbering based on mature polypeptide chain) in more detail by conservative mutation to arginine (K301R) and non-conservative mutation to leucine (K301L). To assess specific FIX activity, F9-K301 variants were transiently expressed in HEK293T cells and tested for antigenic FIX levels and chromogenic activity 48 hours post transfection. To assess specific activity in plasma, AAV-mediated gene transfer (1x1010vg/mouse) of F9-K301 variants in hemophilia B knock-out mice (CL57B6) was carried out. In addition, we investigated whether the F9-K301R mutation enhances specific activity in combination with the F9-R338L Padua mutation via site-specific genome integration. Results: Transient transfection of F9-K301 variants in HEK293T cells showed a 25% increase in specific activity with F9-K301R but a 50% reduction in activity with F9-K301L as compared to wild type F9 (WT-F9). Validation of gain-of-function was done by AAV-mediated gene transfer in hemophilia B knock-out mice. Four weeks post injection, plasma FIX antigen levels were similar in mice transduced with either F9-K301R (0.91±0.3 U/ml; N=3), F9-K301L (0.93±0.0 U/ml; N=2) or WT-F9 (0.94±0.19 U/ml; N=4) constructs. Interestingly, specific chromogenic activity in plasma from F9-K301R mice (2.71±0.66 U/ml) was more than 2-fold higher compared to plasma from mice in the WT-F9 cohort (1.25±0.2 U/ml). On the other hand, specific activity in the F9-K301L cohort (0.37±0.07 U/ml) was reduced compared to wild type F9, consistent with a haemophilic phenotype. Next, we investigated whether the F9-K301R mutation enhances activity in combination with the F9-R338L Padua mutation. To do so, we stably expressed wild type FIX (WT-FIX) and three FIX gain-of-function variants (FIX-K301R, FIX-R338L and FIX-K301R/R338L) in HEK293 cells via site-specific genome integration. Interestingly, higher FIX antigen levels were observed in conditioned media from cells (1.5x106) stably expressing FIX-K301R (0.14±0.01 U/ml) FIX-R338L (0.11±0.01 U/ml) and FIX-K301R/R338L (0.10±0.01 U/ml) relative to cells expressing WT-FIX (0.08±0.01 U/ml). Similar to previous results, specific chromogenic activity was more than 2-fold higher in FIX-K301R (1.25±0.08 U/ml) compared to WT-FIX (0.54±0.06 U/ml). In addition, specific activity was higher in FIX-K301R/R338L (7.71±0.35 U/ml) compared to FIX-R338L (6.69±0.32 U/ml), suggesting molecular synergism between both gain-of-function mutations. Ongoing studies are focused on characterizing these recombinant FIX variants in purified and plasma-based activity assays and unraveling the mechanism(s) leading to increased expression/secretion of these gain-of-function variants. Conclusion: In summary, these results show that the K301R mutation enhances catalytic activity of FIX in vitro and in vivo and synergistically enhances activity in combination with the R338L Padua mutation. As such, this gain-of-function mutation could potentially serve to facilitate higher levels of FIX activity in the plasma of Haemophilia B patients following AAV-mediated gene transfer. Disclosures Verhoef: Freeline: Employment, Equity Ownership. Foley:Freeline: Employment, Equity Ownership. Goodale:Freeline: Employment, Equity Ownership. Macrae:Freeline: Employment, Equity Ownership. McIntosh:BioMarin: Patents & Royalties; Freeline: Consultancy, Equity Ownership. Corbau:Freeline: Employment, Equity Ownership. Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Introduction Over recent years, proof-of-concept academic and early phase commercial trials have demonstrated long-term therapeutic benefit of liver directed AAV-mediated gene transfer of human FIX (hFIX). Extending this success to a greater number of haemophilia B patients is hampered by the need for large vector doses required for efficient gene transfer and associated liver toxicity. To improve potency, we focused on developing a novel synthetic capsid, AAVS3, to increase human hepatocyte transduction. Here we present the preclinical in vitro and in vivo evaluation of AAVS3 pseudotyped vectors. In addition to showing strong liver tropism we demonstrate clinically relevant expression levels of hFIX expression in non human primates. Methods Primary human hepatocyte cultures were used to compare transduction efficiency of our novel capsid with AAV5 and AAV8, variants currently in the clinic. To extend this data in vivo, a humanised mouse liver model was utilised. Subsequently, fourteen Rhesus macaques were dosed with AAVS3 pseudotyped FIX vectors, via saphenous vein injection. Levels of hFIX were determined using an ELISA assay that is able to distinguish hFIX protein from the native Rhesus macaque FIX protein. At harvest biodistribution of vector was detected by quantitative PCR. Results We demonstrate that gene transfer efficiency with AAVS3 was up to 10-fold higher in primary human hepatocytes, when compared to vectors pseudotyped with AAV serotype 5 or 8 capsids. In addition, studies in a chimeric human-murine liver model revealed AAVS3 was 6-fold more efficient at transducing human hepatocytes compared with an AAV8 vector. Using two distinct optimised FIX expression cassettes, we demonstrate highly efficient gene transfer in Rhesus macaques. Indeed, AAVS3 FIX vectors injected at clinically relevant doses of 0.93 x1012 to 2.56 x1013 vg/kg achieved mean peak levels of 0.546 and 21.7µg/ml of hFIX respectively (~11% to ~430% of normal levels). Biodistribution studies showed strong liver tropism following peripheral vein administration with minimal distribution to other organs at or below the level of detection. No vector associated lesions or adverse events were observed in the liver. Conclusion Collectively, these data support further evaluation of an AAVS3 pseudotyped vector in humans with the aim of achieving transduction of a larger proportion of hepatocytes using a lower, potentially safer dose of vector than achieved with AAV8. Disclosures Dane: Freeline: Employment. Sheridan:Freeline: Employment, Equity Ownership. Maginn:Freeline: Consultancy, Equity Ownership. Corbau:Freeline: Employment, Equity Ownership. Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy, Patents & Royalties; UniQure: Patents & Royalties.

Introduction: Fabry disease (FD) is an X-linked lysosomal storage disorder resulting from mutations in the gene encoding α-galactosidase A (GLA). It is characterised by the abnormal accumulation of neutral glycosphingolipids (GSL), predominantly Globotriaosylceramide (Gb3), in the lysosomes of multiple cell types including vascular endothelial cells. It is associated with early-onset stroke, cardiomyopathy, and progression to end-stage renal failure. Current standard of care for FD is enzyme replacement therapy but alternatives including allogeneic stem cell transplantation have also been explored. Fabry disease is a good model for liver-targeted gene therapy, as relatively low levels of enzyme correction may suffice to reduce storage. Additionally, a broad therapeutic window exists, and mouse models of FD enabling preclinical proof-of-concept studies are readily available. Here we report the preclinical results of adeno-associated virus (AAV) mediated, liver-targeted gene transfer of a self-complementary (sc) vector encoding a codon-optimised GLA transgene under transcriptional control of a hepatocyte-specific promoter pseudotyped with AAV8 capsid (scAAV2/8-LP1-GLAco). Methods: In this study a single dose of scAAV2/8-LP1-GLAco vectors were administered into the tail vein of homozygote GLA knockout Fabry mice at either 1 month or 3 months of age. Two doses were tested for each age group; 2x1012 vg/kg and 2x1013 vg/kg, and an additional age-matched group left as untreated to serve as an experimental control for the effects of treatment. Mice were followed in the treatment for up to 11 months before sacrificing at 11 or 14 months of age. To assess the kinetics and durability of transgene expression, plasma GLA levels were measured at various time intervals post injection. Results: Long-term follow-up of Fabry mice injected with scAAV2/8-LP1-GLAco vector demonstrated sustained, durable levels of plasma GLA throughout the typical lifespan of a mouse. Plasma GLA activity levels at 10 or 11-months post vector administration to 1 and 3-month-old mice respectively, demonstrated a dose response from 2 x 1012 to 2 x 1013 vg/kg of >1 log increase in expression. These GLA activity levels represent supra-physiological expression (x 4000 of normal levels) which resulted in uptake of GLA from the bloodstream into kidney and cardiac tissues leading to dose-dependent clearance of lipid deposits in these tissues as analysed by electron microscopy. Most importantly, sustained expression of GLA resulted in complete normalisation of Gb3 and lyso-Gb3 levels in plasma and liver tissues as quantified by mass spectrometry. Interestingly, these preclinical studies in Fabry mice at the age of 1 and 3 months demonstrated not only prevention but also reversal of lysosomal storage deposits through liver-specific expression of GLA. Finally, in these in vivo mouse studies no adverse clinical signs were observed, despite the increased plasma exposure to GLA. Conclusions: Collectively, these data provide strong evidence that our liver-directed AAV-mediated gene therapy approach holds considerable therapeutic potential for the treatment of Fabry disease. We anticipate that a single dose IV procedure will pose minimal burden to Fabry patients and will be a viable alternative to biweekly enzyme infusions, potentially reducing treatment-related morbidity whislt improving patient quality of life and potentially providing them with a functional long-term cure. Disclosures Kia: Freeline: Employment, Equity Ownership. McIntosh:Freeline: Consultancy. Hosseini:Freeline: Employment, Equity Ownership. Sheridan:Freeline: Employment, Equity Ownership. Corbau:Freeline: Employment, Equity Ownership. Nathwani:BioMarin: Consultancy, Patents & Royalties; UniQure: Patents & Royalties; Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.