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Fabry disease is an X-linked lysosomal storage disorder caused by loss of alpha-galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of glycosphingolipids in multiple cells and tissues. FLT190, an investigational gene therapy, is currently being evaluated in a Phase 1/2 clinical trial in patients with Fabry disease (NCT04040049). FLT190 consists of a potent, synthetic capsid (AAVS3) containing an expression cassette with a codon-optimized human GLA cDNA under the control of a liver-specific promoter FRE1 (AAV2/S3-FRE1-GLAco). For mouse studies FLT190 genome was pseudotyped with AAV8 for efficient transduction. Preclinical studies in a murine model of Fabry disease (Gla-deficient mice), and non-human primates (NHPs) showed dose-dependent increases in plasma α-Gal A with steady-state observed 2 weeks following a single intravenous dose. In Fabry mice, AAV8-FLT190 treatment resulted in clearance of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) in plasma, urine, kidney, and heart; electron microscopy analyses confirmed reductions in storage inclusion bodies in kidney and heart. In NHPs, α-Gal A expression was consistent with the levels of hGLA mRNA in liver, and no FLT190-related toxicities or adverse events were observed. Taken together, these studies demonstrate preclinical proof-of-concept of liver-directed gene therapy with FLT190 for the treatment of Fabry disease.

Adeno-associated virus (AAV) gene therapy has the potential to functionally cure hemophilia B by restoring factor (F)IX concentrations into the normal range. Next-generation AAV therapies express a naturally occurring gain-of-function FIX variant, FIX-Padua (R338L-FIX), that increases FIX activity (FIX:C) by approximately eightfold compared with wild-type FIX (FIX-WT). Previous studies have shown that R338L-FIX activity varies dramatically across different clinical FIX:C assays, which complicates the monitoring and management of patients. To better understand mechanisms that contribute to R338L-FIX assay discrepancies, we characterized the performance of R338L-FIX in 13 1-stage clotting assays (OSAs) and 2 chromogenic substrate assays (CSAs) in a global field study. This study produced the largest R338L-FIX assay dataset to date and confirmed that clinical FIX:C assay results vary over threefold. Both phospholipid and activating reagents play a role in OSA discrepancies. CSA generated the most divergent FIX:C results. Manipulation of FIX:C CSA kits demonstrated that specific activity gains for R338L-FIX were most profound at lower FIX:C concentrations and that these effects were enhanced during the early phases of FXa generation. Supplementing FX into CSA had the effect of dampening FIX-WT activity relative to R338L-FIX activity, suggesting that FX impairs WT tenase formation to a greater extent than R338L-FIX tenase. Our data describe the scale of R338L-FIX assay discrepancies and provide insights into the causative mechanisms that will help establish best practices for the measurement of R338L-FIX activity in patients after gene therapy.

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.

Gaucher disease (GD) is characterised by the deposition of glucocerebroside in cells of the macrophage-monocyte system caused by impaired production of the enzyme beta-glucocerebrosidase (GCase). Over the past 20 years, enzymatic replacement therapy (ERT) was developed as the standard of care for GD. However, since it requires continuous treatment (every other week infusions), it results in a high cumulative cost for this type of therapy, and more importantly in a significant patient treatment burden. Over the same period, gene therapy has emerged as a very promising avenue of treatment for various monogenic genetic disorders, allowing sustained levels of transgene expression upon a single treatment. We hypothesised that liver-directed gene therapy could be used to ameliorate GD disease. For this purpose, we used an adeno-associated viral vector (AAV) to drive the expression of human GBA gene in the liver of mice. Four weeks after a single injection we observed a steady and dose-dependent elevation of GCase in the bloodstream of treated mice. Upon tissue analysis, noticeable uptake of human GCase was observed in organs affected in GD such as spleen, lung and bone marrow. Marker analysis performed in spleen tissue showed positive staining for human GCase in macrophages. Further improvement in our AAV constructs, obtained by applying rational design and codon optimisation algorithms, has allowed us to achieve an additional five-fold increase in GCase circulating in the bloodstream, resulting in increased uptake by macrophages. Overall, these data show that upon a single injection of a liver-expression-directed AAV vector it is possible to achieve elevation of GCase in the bloodstream that results in greater GCase bioavailability compared to current ERT. Therefore, this approach offers enhanced therapeutic potential for the treatment of GD.

Fabry disease is an X-linked lysosomal storage disorder (LSD) resulting from mutations in the gene encoding for 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. We examined the therapeutic efficacy of GLA gene therapy in Fabry mice using recombinant adeno-associated virus (rAAV) vectors, directed by the liver-specific promoter (FRE1). In this study, we showed that a single intravenous (IV) injection of rAAV8-FRE1-GLAco (2x1012 vg/kg) achieved supraphysiological levels of plasma GLA activity up to 1061-fold of normal with concomitant correction of lysosomal storage pathology in multiple key organs in a Fabry mouse model. Animals injected with our novel codon-optimised (“GLAco”) GLA construct exhibited a rapid elevation in plasma GLA activity levels reaching a peak level by 4 weeks post-injection, and this level of expression was maintained for the duration of the study (14 weeks). Furthermore, mass spectrometry (LC-MC/MS) analysis of GSL extract from different tissues provided proof of exposure and storage clearance in various key organs, which is indicative of metabolic cross-correction of liver-derived GLA enzyme. Gb3 levels in the plasma were reduced by 91% (p=0.008) in the kidney by 64% (p=0.045) in the heart by 98% (p

FLT3 and PDGFR tyrosine kinases are important targets for therapy of different types of leukemia. Several FLT3/PDGFR inhibitors are currently under clinical investigation for combination with standard therapy for treatment of acute myeloid leukemia (AML), however these agents only induce partial remission and development of resistance has been reported. In this work we describe the identification of potent and novel dual FLT3/PDGFR inhibitors that resulted from our efforts to screen a library of 25,607 small molecules against the FLT3 dependent cell line MOLM-13 and the PDGFR dependent cell line EOL-1. This effort led to the identification of five compounds that were confirmed to be active on additional FLT3 dependent cell lines (cellular EC50 values between 35 and 700 nM), while having no significant effect on 24 other tyrosine kinases.

Objective: Spinocerebellar ataxia type 1 is an autosomal dominant fatal neurodegenerative disease caused by a polyglutamine expansion in the coding region of ATXN1. We showed previously that partial suppression of mutant ataxin-1 (ATXN1) expression, using virally-expressed RNAi triggers, could prevent disease symptoms in a transgenic mouse model and a knock in mouse model of the disease, using a single dose of virus. Here, we set out to test if RNAi triggers targeting ATXN1 could not only prevent, but also reverse disease readouts when delivered after symptom onset. Methods: We administered recombinant adeno-associated virus (rAAV) expressing miS1, an artificial miRNA targeting human ATXN1 mRNA (rAAV.miS1) to a mouse model of SCA1 (B05 mice). Viruses were delivered prior to or after symptom onset at multiple doses. Control B05 mice were treated with rAAVs expressing a control artificial miRNA, or with saline. Animal behavior, molecular phenotypes, neuropathology and magnetic resonance spectroscopy were done on all groups and data were compared to wildtype littermates. Results: We found that SCA1 phenotypes could be reversed by partial suppression of human mutant ATXN1 mRNA by rAAV.miS1 when delivered after symptom onset. We also identified the therapeutic range of rAAV.miS1 that could prevent or reverse disease readouts. Interpretation: SCA1 disease may be reversible by RNAi therapy, and the doses required for advancing this therapy to humans are delineated. This article is protected by copyright. All rights reserved.

The hepatitis C virus (HCV) chronically infects 2% of the world population and effective treatment is limited by long duration and significant side-effects. Here, we describe a novel drug, intended as a “single-shot ” therapy, which expresses three short hairpin RNAs (shRNAs) that simultaneously target multiple conserved regions of the HCV genome as confirmed in vitro by knockdown of an HCV replicon system. Using a recombinant adeno-associated virus (AAV) serotype 8 vector for delivery, comprehensive transduction of hepatocytes was achieved in vivo in a nonhuman primate (NHP) model following a single intravenous injection. However, dose ranging studies performed in 13 NHP resulted in high-expression levels of shRNA from wild-type (wt) Pol III promoters and dose-dependent hepatocellular toxicity, the first demonstration of shRNA-related toxicity in primates, establishing that the hepatotoxicity arises from highly conserved features of the RNA interference (RNAi) pathway. In the second generation drug, each promoter was re-engineered to reduce shRNA transcription to levels that circumvent toxicity but still inhibit replicon activity. In vivo testing of this modified construct in 18 NHPs showed conservation of hepatocyte transduction but complete elimination of hepatotoxicity, even with sustained shRNA expression for 50 days. These data support progression to a clinical study for treatment of HCV infection.

We have screened 47 locked nucleic acid (LNA) antisense oligonucleotides (ASOs) targeting conserved (>95% homology) sequences in the hepatitis C virus (HCV) genome using the subgenomic HCV replicon assay and generated both antiviral (50% effective concentration [EC 50 ]) and cytotoxic (50% cytotoxic concentration [CC 50 ]) dose-response curves to allow measurement of the selectivity index (SI). This comprehensive approach has identified an LNA ASO with potent antiviral activity (EC 50 = 4 nM) and low cytotoxicity (CC 50 >880 nM) targeting the 25- to 40-nucleotide region (nt) of the HCV internal ribosome entry site (IRES) containing the distal and proximal miR-122 binding sites. LNA ASOs targeting previously known accessible regions of the IRES, namely, loop III and the initiation codon in loop IV, had poor SI values. We optimized the LNA ASO sequence by performing a 1-nucleotide walk through the 25- to 40-nt region and show that the boundaries for antiviral efficacy are extremely precise. Furthermore, we have optimized the format for the LNA ASO using different gapmer and mixomer patterns and show that RNase H is required for antiviral activity. We demonstrate that RNase H-refractory ASOs targeting the 25- to 40-nt region have no antiviral effect, revealing important regulatory features of the 25- to 40-nt region and suggesting that RNase H-refractory LNA ASOs can act as potential surrogates for proviral functions of miR-122. We confirm the antisense mechanism of action using mismatched LNA ASOs. Finally, we have performed pharmacokinetic experiments to demonstrate that the LNA ASOs have a very long half-life (>5 days) and attain hepatic maximum concentrations >100 times the concentration required for in vitro antiviral activity.

Lersivirine is a potent non-nucleoside reverse transcriptase inhibitor with exceptional mutant resilience. Here, we compare the pharmacological and pharmacokinetic profile of lersivirine with its pyrazole and imidazole isomers and briefly explore the profile of these series. This work establishes lersivirine as the outstanding molecule in this set.

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.

The nonnucleoside reverse transcriptase inhibitors (NNRTIs) are key components of highly active antiretroviral therapy (HAART) for the treatment of human immunodeficiency virus type 1 (HIV-1). A major problem with the first approved NNRTIs was the emergence of mutations in the HIV-1 reverse transcriptase (RT), in particular K103N and Y181C, which led to resistance to the entire class. We adopted an iterative strategy to synthesize and test small molecule inhibitors from a chemical series of pyrazoles against wild-type (wt) RT and the most prevalent NNRTI-resistant mutants. The emerging candidate, lersivirine (UK-453,061), binds the RT enzyme in a novel way (resulting in a unique resistance profile), inhibits over 60% of viruses bearing key RT mutations, with 50% effective concentrations (EC50s) within 10-fold of those for wt viruses, and has excellent selectivity against a range of human targets. Altogether lersivirine is a highly potent and selective NNRTI, with excellent efficacy against NNRTI-resistant viruses.

A major problem associated with non-nucleoside reverse transcriptase inhibitors (NNRTIs) for the treatment of HIV is their vulnerability to mutations in the allosteric binding site of reverse transcriptase that can result in the development of a resistant virus. Herein we present the optimization of a series of 5-aryloxy imidazoles, which possess a balanced pharmacological profile against both wild-type enzyme and the clinically relevant mutations K103N and Y181C. Subtle structural changes were used to probe structure-activity relationships relating to both potency and metabolic stability, which led to an imidazole derivative with an impressive overall profile.

This article describes the automation of an in vitro cell-based fusion assay for the identification of novel inhibitors of receptor mediated HIV-1 entry. The assay utilises two stable cell lines: one expressing CD4, CCR5 and an LTR-promoter/beta-galactosidase reporter construct, and the other expressing gp160 and tat. Accumulation of beta-galactosidase can only occur following fusion of these two cell lines via the gp160 and receptor mediators, as this event facilitates the transfer of the tat transcription factor between the two cell types. Although similar cell fusion systems have been described previously, they have not met the requirements for HTS due to complexity, throughput and reagent cost. The assay described in this article provides significant advantage, as (a) no transfection/infection events are required prior to the assay, reducing the potential for variability, (b) cells are mixed in solution, enhancing fusion efficiency compared to adherent cells, (c) miniaturization to low volume enables screening in 384-well plates; and (d) online cell dispensing facilitates automated screening. This assay has been employed to screen approximately 650,000 compounds in a singleton format. The data demonstrate that the assay is robust, with a Z' consistently above 0.6, which compares favourably with less complex biochemical assays.

Mutations in the gene encoding Retinal Pigment Epithelium-Specific Protein 65 (RPE65) cause impaired vision from an early age and eventually total vision loss later in life. Recent work by our group and others demonstrated the use of SPK-RPE65, an adeno-associated viral (AAV) vector, to deliver the gene stably in small and large animal models, while Phase 1 and Phase 3 clinical trials with SPK-RPE65 showed promising results (Maguire et al., 2008; Maguire et al., 2009; Simonelli et al., 2010; Maguire, AAO 2015). The Phase 3 trial was an open-label, randomized study using a subretinally-delivered AAV2 vector to augment the RPE65 gene. Thirty one subjects were enrolled from centers at The Children's Hospital of Philadelphia or University of Iowa. Twenty of the 21 intervention group subjects received 1.5E11 vg non-simultaneous injections to each eye. In addition to a separate efficacy assessment that measured the primary and secondary endpoints for the trial, an independent safety study was performed that included previously established immunological assays designed to monitor cellular immune responses. Two assays were subjected to further validation in-house and used for immune analysis of clinical samples obtained from all intervention subjects. An Enzyme-Linked Immunosorbent Assay (ELISA), capable of detecting a titer of at least 1.55 ug/mL anti-AAV2-capsid human IgG, was performed on 21 subjects covering up to 4 timepoints (baseline prior to injection, day 30, day 90 and 1 year). To better evaluate the results, the subjects were placed into 6 categories based on their antibody titer profile (Table 1Table 1).Table 1Anti-AAV2 Profile of Intervention Group (n=21)Number of SubjectsAnti AAV Titer Range (ug/mL)Anti-AAV2 Profile7

Minute virus of mice NS1, an 83-kDa mainly nuclear phosphoprotein, is the only viral nonstructural protein required in all cell types and it is involved in multiple processes necessary for virus propagation. The diversity of functions assigned to NS1, together with the variation of its complex phosphorylation pattern during infection, suggested that the various activities of NS1 could be regulated by distinct phosphorylation events. So far, it has been demonstrated that NS1 replicative functions, in particular, DNA-unwinding activities, are regulated by protein kinase C (PKC), as exemplified by the modulation of NS1 helicase activity by PKCλ phosphorylation. In order to determine further impact of phosphorylation on NS1 functions, including the induction of cytopathic effects, a mutational approach was pursued in order to produce NS1 variants harboring amino acid substitutions at candidate PKC target residues. Besides the determination of two additional in vivo phosphorylation sites in NS1, this mutagenesis allowed the segregation of distinct NS1 functions from one another, generating NS1 variants with a distinct activity profile. Thus, we obtained NS1 mutants that were fully proficient for trans activation of the viral P38 promoter, while being impaired in their replicative functions. Moreover, the alterations of specific PKC phosphorylation sites gave rise to NS1 polypeptides that exerted reduced cytotoxicity, leading to sustained gene expression, while keeping functions necessary for progeny virus production, i.e., viral DNA replication and activation of the capsid gene promoter. These data suggested that in the course of a viral infection, NS1 may undergo a shift from productive to cytotoxic functions as a result of a phosphorylation-dependent regulation.

The major nonstructural protein of parvovirus MVMp, NS1, is an 83-kDa nuclear phosphoprotein which exerts a variety of functions during a viral infection. These multiple tasks range from its major involvement in viral DNA amplification and promoter regulation to the cytotoxic action on the host cell. Since these most divergent functions are exerted in an orderly fashion, it has been proposed that NS1 is regulated by posttranslational modifications, in particular phosphorylation. So far it has been shown that the capacity of NS1 for initiation of replication is regulated in vitro by phosphorylation through members of the protein kinase C family, most likely as a result of control of the DNA unwinding activity (J. P. F. Nuesch et al., 1998, J. Virol. 72, 9966–9977). To substantiate these in vitro findings in vivo, we investigated NS1 phosphorylation during an MVMp infection in a natural host cell, A9 fibroblasts, with reference to characteristic features of the virus cycle. The NS1 phosphorylation pattern was found to change throughout the infection, raising the possibility that distinct tasks of NS1 might be achieved through differential phosphorylation of the polypeptide. In addition, we present in vivo evidence that a phosphorylated form of NS1 is able to initiate viral DNA replication and becomes covalently attached to replicated DNA. Moreover, NS1 was found to be phosphorylated in vivo within the helicase domain, showing alignment with at least one phosphopeptide generated by an “activating” kinase in vitro. These data suggest that phosphorylation-mediated regulation of NS1 for replicative functions as observed in vitro may also take place during a natural virus infection.

NS1, the major nonstructural protein of the parvovirus minute virus of mice, is a multifunctional phosphoprotein which is involved in cytotoxicity, transcriptional regulation, and initiation of viral DNA replication. For coordination of these various functions during virus propagation, NS1 has been proposed to be regulated by posttranslational modifications, in particular phosphorylation. Recent in vitro studies (J. P. F. Nüesch, R. Corbau, P. Tattersall, and J. Rommelaere, J. Virol. 72:8002–8012, 1998) provided evidence that distinct NS1 activities, notably the intrinsic helicase function, are modulated by the phosphorylation state of the protein. In order to study the dependence of the initiation of viral DNA replication on NS1 phosphorylation and to identify the protein kinases involved, we established an in vitro replication system that is devoid of endogenous protein kinases and is based on plasmid substrates containing the minimal left-end origins of replication. Cellular components necessary to drive NS1-dependent rolling-circle replication (RCR) were freed from endogenous serine/threonine protein kinases by affinity chromatography, and the eukaryotic DNA polymerases were replaced by the bacteriophage T4 DNA polymerase. While native NS1 (NS1 P ) supported RCR under these conditions, dephosphorylated NS1 (NS1 O ) was impaired. Using fractionated HeLa cell extracts, we identified two essential protein components which are able to phosphorylate NS1 O , are enriched in protein kinase C (PKC), and, when present together, reactivate NS1 O for replication. One of these components, containing atypical PKC, was sufficient to restore NS1 O helicase activity. The requirement of NS1 O reactivation for characteristic PKC cofactors such as Ca 2+ /phosphatidylserine or phorbol esters strongly suggests the involvement of this protein kinase family in regulation of NS1 replicative functions in vitro.

The promoter of the thyroid hormone receptor alpha gene (c-erbA-1) is activated by the nonstructural protein 1 (NS1) of parvovirus minute virus of mice (prototype strain [MVMp]) in ras-transformed FREJ4 cells that are permissive for lytic MVMp replication. This stimulation may be related to the sensitivity of host cells to MVMp, as it does not take place in parental FR3T3 cells, which are resistant to the parvovirus killing effect. The analysis of a series of deletion and point mutants of the c-erbA-1 promoter led to the identification of an upstream region that is necessary for NS1-driven transactivation. This sequence harbors a putative hormone-responsive element and is sufficient to render a minimal promoter NS1 inducible in FREJ4 but not in FR3T3 cells, and it is involved in distinct interactions with proteins from the respective cell lines. The NS1-responsive element of the c-erbA-1 promoter bears no homology with sequences that were previously reported to be necessary for NS1 DNA binding and transactivation. Altogether, our data point to a novel, cell-specific mechanism of promoter activation by NS1.

Lersivirine is a potent non-nucleoside reverse transcriptase inhibitor with exceptional mutant resilience. Here, we compare the pharmacological and pharmacokinetic profile of lersivirine with its pyrazole and imidazole isomers and briefly explore the profile of these series. This work establishes lersivirine as the outstanding molecule in this set.

We prepared three discreet cohorts of potent non-nucleoside HIV reverse transcriptase inhibitors (NNRTIs) based on the recently reported 3-cyanophenoxypyrazole lead 3. Several of these compounds displayed very promising anti-HIV activity in vitro, safety, pharmacokinetic and pharmaceutical profiles. We describe our analysis and conclusions leading to the selection of alcohol 5 (UK-453,061, lersivirine) for clinical development.

Our efforts to reduce overall lipophilicity and increase ligand-lipophilicity efficiency (LLE) by modification of the 3- and 5-substituents of pyrazole 1, a novel non-nucleoside HIV reverse transcriptase inhibitor (NNRTI) prototype were unsuccessful. In contrast replacement of the substituted benzyl group with corresponding phenylthio or phenoxy groups resulted in marked improvements in potency, ligand efficiency (LE) and LLE.

The design and synthesis of a novel series of non-nucleoside HIV reverse transcriptase inhibitors (NNRTIs) based on a pyrazole template is described. These compounds are active against wild type reverse transcriptase (RT) and retain activity against clinically important mutants.

Existing HCV protease inhibitors fall into two categories: reversible and non-covalent, such as BILN-2061, and covalent and reversible, exemplified by SCH-503034 and VX-950. In this work, the characterization of the kinetics of these three inhibitors is presented. SCH-503034 and VX-950 initially bind to the genotype 1b HCV NS3/4A protease to form a low affinity complex, with K(i) values of 5 and 5.8 microM respectively. The ability of those two compounds to form a second covalent complex (EI) results in a potency increase, with overall K(i) values of 20 and 45 nM, respectively. The increase in potency can be explained by their slow dissociation rate, forming complexes with half-lives of 2 h (VX-950) and 5 h (SCH-503034). Although BILN-2061 has been described as a fast reversible, non-covalent inhibitor, our results show a slow binding two-step mechanism. Contrary to SCH-503034 and VX-950, BILN-2061 can form a high affinity first complex with a K(i) value of 3.9 nM, and an overall K(i) of 0.14 nM. The half-life of the BILN-2061 EI complex is shorter (t(1/2) approximately 0.7 h) than that of the other two compounds. The potency of these compounds is genotype dependent, and a kinetic analysis using NS3/4A from genotype 3a indicates that the loss of potency of SCH-503034 and VX-950 relative to genotype 1 is mainly due to the slow on-rate and faster off-rate for the formation of the EI complex. In the case of BILN-2061, a better fit is obtained using a one-step model, indicating that the loss of potency is due to an increase in the off-rate of the EI complex.

Infection of eukaryotic cells by enveloped viruses requires the merging of viral and cellular membranes. Highly specific viral surface glycoproteins, named fusion proteins, catalyze this reaction by overcoming inherent energy barriers. Hepatitis C virus (HCV) is an enveloped virus that belongs to the genus Hepacivirus of the family Flaviviridae . Little is known about the molecular events that mediate cell entry and membrane fusion for HCV, although significant progress has been made due to recent developments in infection assays. Here, using infectious HCV pseudoparticles (HCVpp), we investigated the molecular basis of HCV membrane fusion. By searching for classical features of fusion peptides through the alignment of sequences from various HCV genotypes, we identified six regions of HCV E1 and E2 glycoproteins that present such characteristics. We introduced conserved and nonconserved amino acid substitutions in these regions and analyzed the phenotype of HCVpp generated with mutant E1E2 glycoproteins. This was achieved by (i) quantifying the infectivity of the pseudoparticles, (ii) studying the incorporation of E1E2 and their capacity to mediate receptor binding, and (iii) determining their fusion capacity in cell-cell and liposome/HCVpp fusion assays. We propose that at least three of these regions (i.e., at positions 270 to 284, 416 to 430, and 600 to 620) play a role in the membrane fusion process. These regions may contribute to the merging of viral and cellular membranes either by interacting directly with lipid membranes or by assisting the fusion process through their involvement in the conformational changes of the E1E2 complex at low pH.

Minute virus of mice NS1 protein is a multifunctional phosphoprotein endowed with a variety of enzymatic and regulatory activities necessary for progeny virus particle production. To regulate all of its different functions in the course of a viral infection, NS1 has been proposed to be modulated by posttranslational modifications, in particular, phosphorylation. Indeed, it was shown that the NS1 phosphorylation pattern is altered during the infectious cycle and that the biochemical profile of the protein is dependent on the phosphorylation state of the polypeptide. Moreover, in vitro approaches have identified members of the protein kinase C (PKC) family, in particular, atypical PKC, as regulators of viral DNA replication through the phosphorylation of NS1 residues T435 and S473, thereby activating the protein for DNA unwinding activities. In order to substantiate these findings in vivo, we produced NS1 in the presence of a dominant-negative PKCλ mutant and characterized the purified protein in vitro. The NS1 protein produced under these conditions was found to be only partially phosphorylated and as a consequence to be deficient for viral DNA replication. However, it could be rescued for this viral function by treatment with recombinant activated PKCλ. Our data clearly demonstrate that NS1 is a target for PKCλ phosphorylation in vivo and that this modification is essential for the helicase activity of the viral polypeptide. In addition, the phosphorylation of NS1 at residues T435 and S473 appeared to occur mainly in the nucleus, providing further evidence for the involvement of PKCλ which, unlike PKCζ, accumulates in the nuclear compartment of infected cells.

TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034-encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5' RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC).Molecular Therapy-Nucleic Acids (2014) 3, e145; doi:10.1038/mtna.2013.73; published online 4 February 2014.

NS1, the 83-kDa major nonstructural protein of minute virus of mice (MVM), is a multifunctional nuclear phosphoprotein which is required in a variety of steps during progeny virus production, early as well as late during infection. NS1 is the initiator protein for viral DNA replication. It binds specifically to target DNA motifs; has site-specific single-strand nickase, intrinsic ATPase, and helicase activities; trans regulates viral and cellular promoters; and exerts cytotoxic stress on the host cell. To investigate whether these multiple activities of NS1 depend on posttranslational modifications, in particular phosphorylation, we expressed His-tagged NS1 in HeLa cells by using recombinant vaccinia viruses, dephosphorylated it at serine and threonine residues with calf intestine alkaline phosphatase, and compared the biochemical activities of the purified un(der)phosphorylated (NS1 O ) and the native (NS1 P ) polypeptides. Biochemical analyses of replicative functions of NS1 O revealed a severe reduction of intrinsic helicase activity and, to a minor extent, of ATPase and nickase activities, whereas its affinity for the target DNA sequence [ACCA] 2–3 was enhanced compared to that of NS1 P . In the presence of endogenous protein kinases found in replication extracts, NS1 O showed all functions necessary for resolution and replication of the 3′ dimer bridge, indicating reactivation of NS1 O by rephosphorylation. Partial reactivation of the helicase activity was found as well when NS1 O was incubated with protein kinase C.