AAV Gene Augmentation of Truncated Complement Factor H Differentially Rescues Ocular Complement Dysregulation in a Mouse Model.

Purpose: Complement dysregulation in the eye has been implicated in the pathogenesis of age-related macular degeneration (AMD), and genetic variants of complement factor H (CFH) are strongly associated with AMD risk. We therefore aimed to untangle the role of CFH and its splice variant, factor H-like 1 (FHL-1), in ocular complement regulation derived from local versus circulating sources. We assessed the therapeutic efficacy of adeno-associated viruses (AAVs) expressing human FHL-1 and a truncated version of CFH (tCFH), which retains the functional N- and C-terminal ends of the CFH protein, in restoring the alternative complement pathway in Cfh-/- mouse eyes and plasma. Methods: Using Cfh-/- mice as a model of complement dysregulation, AAV vectors expressing tCFH or FHL-1 were injected subretinally or via tail vein, and the efficacy of the constructs was evaluated. Results: Following subretinal injections, tCFH expression rescued factor B (FB) retention in the eye, but FHL-1 expression did not. By contrast, both constructs restored FB detection in plasma following tail vein injections. Both tCFH and FHL-1 proteins accumulated in the posterior eyecup from the circulation following liver transduction; however, neither was able to significantly regulate local ocular complement. Conclusions: Our findings demonstrate that the C-terminus of human CFH is necessary for complement regulation in the murine eye. Furthermore, exogenous CFH must be synthesized locally to maximize complement regulation in the retina. These findings establish a critical foundation for development of CFH augmentation-based gene therapies for the eye.

Preclinical studies in support of phase I/II clinical trials to treat GUCY2D-associated Leber congenital amaurosis.

Mutations in GUCY2D are associated with severe early-onset retinal dystrophy, Leber congenital amaurosis type 1 (LCA1), a leading cause of blindness in children. Despite a high degree of visual disturbance stemming from photoreceptor dysfunction, patients with LCA1 largely retain normal photoreceptor structure, suggesting that they are good candidates for gene replacement therapy. The purpose of this study was to conduct the preclinical and IND-enabling experiments required to support clinical application of AAV5-hGRK1-GUCY2D in patients harboring biallelic recessive mutations in GUCY2D. Preclinical studies were conducted in mice to evaluate the effect of vector manufacturing platforms and transgene species on the therapeutic response. Dose-ranging studies were conducted in cynomolgus monkeys to establish the minimum dose required for efficient photoreceptor transduction. Good laboratory practice (GLP) studies evaluated systemic biodistribution in rats and toxicology in non-human primates (NHPs). These results expanded our knowledge of dose response for an AAV5-vectored transgene under control of the human rhodopsin kinase (hGRK1) promoter in NHPs with respect to photoreceptor transduction and safety and, in combination with the rat biodistribution and mouse efficacy studies, informed the design of a first-in-human clinical study in patients with LCA1.

Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases.

Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.

Rationally designed AAV2 and AAVrh8R capsids provide improved transduction in the retina and brain.

The successful application of adeno-associated virus (AAV) gene delivery vectors as a therapeutic paradigm will require efficient gene delivery to the appropriate cells in affected organs. In this study, we utilized a rational design approach to introduce modifications to the AAV2 and AAVrh8R capsids and the resulting variants were evaluated for transduction activity in the retina and brain. The modifications disrupted either capsid/receptor binding or altered capsid surface charge. Specifically, we mutated AAV2 amino acids R585A and R588A, which are required for binding to its receptor, heparan sulfate proteoglycans, to generate a variant referred to as AAV2-HBKO. In contrast to parental AAV2, the AAV2-HBKO vector displayed low-transduction activity following intravitreal delivery to the mouse eye; however, following its subretinal delivery, AAV2-HBKO resulted in significantly greater photoreceptor transduction. Intrastriatal delivery of AAV2-HBKO to mice facilitated widespread striatal and cortical expression, in contrast to the restricted transduction pattern of the parental AAV2 vector. Furthermore, we found that altering the surface charge on the AAVrh8R capsid by modifying the number of arginine residues on the capsid surface had a profound impact on subretinal transduction. The data further validate the potential of capsid engineering to improve AAV gene therapy vectors for clinical applications.

Universal Method for the Purification of Recombinant AAV Vectors of Differing Serotypes.

The generation of clinical good manufacturing practices (GMP)-grade adeno-associated virus (AAV) vectors requires purification strategies that support the generation of vectors of high purity, and that exhibit a good safety and efficacy profile. To date, most reported purification schemas are serotype dependent, requiring method development for each AAV gene therapy product. Here, we describe a platform purification process that is compatible with the purification of multiple AAV serotypes. The method generates vector preparations of high purity that are enriched for capsids with full vector genomes, and that minimizes the fractional content of empty capsids. The two-column purification method, a combination of affinity and ion exchange chromatographies, is compatible with a range of AAV serotypes generated by either the transient triple transfection method or the more scalable producer cell line platform. In summary, the adaptable purification method described can be used for the production of a variety of high-quality AAV vectors suitable for preclinical testing in animal models of diseases.

AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.

Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

Intravitreous injection of AAV2-sFLT01 in patients with advanced neovascular age-related macular degeneration: a phase 1, open-label trial.

BACKGROUND: Long-term intraocular injections of vascular endothelial growth factor (VEGF)-neutralising proteins can preserve central vision in many patients with neovascular age-related macular degeneration. We tested the safety and tolerability of a single intravitreous injection of an AAV2 vector expressing the VEGF-neutralising protein sFLT01 in patients with advanced neovascular age-related macular degeneration. METHODS: This was a phase 1, open-label, dose-escalating study done at four outpatient retina clinics in the USA. Patients were assigned to each cohort in order of enrolment, with the first three patients being assigned to and completing the first cohort before filling positions in the following treatment groups. Patients aged 50 years or older with neovascular age-related macular degeneration and a baseline best-corrected visual acuity score of 20/100 or less in the study eye were enrolled in four dose-ranging cohorts (cohort 1, 2 × 108 vector genomes (vg); cohort 2, 2 × 109 vg; cohort 3, 6 × 109 vg; and cohort 4, 2 × 1010 vg, n=3 per cohort) and one maximum tolerated dose cohort (cohort 5, 2 × 1010 vg, n=7) and followed up for 52 weeks. The primary objective of the study was to assess the safety and tolerability of a single intravitreous injection of AAV2-sFLT01, through the measurement of eye-related adverse events. This trial is registered with ClinicalTrials.gov, number NCT01024998. FINDINGS: 19 patients with advanced neovascular age-related macular degeneration were enrolled in the study between May 18, 2010, and July 14, 2014. All patients completed the 52-week trial period. Two patients in cohort 4 (2 × 1010 vg) experienced adverse events that were possibly study-drug related: pyrexia and intraocular inflammation that resolved with a topical steroid. Five of ten patients who received 2 × 1010 vg had aqueous humour concentrations of sFLT01 that peaked at 32·7-112·0 ng/mL (mean 73·7 ng/mL, SD 30·5) by week 26 with a slight decrease to a mean of 53·2 ng/mL at week 52 (SD 17·1). At baseline, four of these five patients were negative for anti-AAV2 serum antibodies and the fifth had a very low titre (1:100) of anti-AAV2 antibodies, whereas four of the five non-expressers of sFLT01 had titres of 1:400 or greater. In 11 of 19 patients with intraretinal or subretinal fluid at baseline judged to be reversible, six showed substantial fluid reduction and improvement in vision, whereas five showed no fluid reduction. One patient in cohort 5 showed a large decrease in vision between weeks 26 and 52 that was not thought to be vector-related. INTERPRETATION: Intravitreous injection of AAV2-sFLT01 seemed to be safe and well tolerated at all doses. Additional studies are needed to identify sources of variability in expression and anti-permeability activity, including the potential effect of baseline anti-AAV2 serum antibodies. FUNDING: Sanofi Genzyme, Framingham, MA, USA.

Soluble FLT1 Gene Therapy Alleviates Brain Arteriovenous Malformation Severity.

BACKGROUND AND PURPOSE: Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current therapies are associated with high morbidities. Excessive vascular endothelial growth factor has been implicated in bAVM pathophysiology. Because soluble FLT1 binds to vascular endothelial growth factor with high affinity, we tested intravenous delivery of an adeno-associated viral vector serotype-9 expressing soluble FLT1 (AAV9-sFLT1) to alleviate the bAVM phenotype. METHODS: Two mouse models were used. In model 1, bAVM was induced in R26CreER;Eng2f/2f mice through global Eng gene deletion and brain focal angiogenic stimulation; AAV2-sFLT02 (an AAV expressing a shorter form of sFLT1) was injected into the brain at the time of model induction, and AAV9-sFLT1, intravenously injected 8 weeks after. In model 2, SM22αCre;Eng2f/2f mice had a 90% occurrence of spontaneous bAVM at 5 weeks of age and 50% mortality at 6 weeks; AAV9-sFLT1 was intravenously delivered into 4- to 5-week-old mice. Tissue samples were collected 4 weeks after AAV9-sFLT1 delivery. RESULTS: AAV2-sFLT02 inhibited bAVM formation, and AAV9-sFLT1 reduced abnormal vessels in model 1 (GFP versus sFLT1: 3.66±1.58/200 vessels versus 1.98±1.29, P<0.05). AAV9-sFLT1 reduced the occurrence of bAVM (GFP versus sFLT1: 100% versus 36%) and mortality (GFP versus sFLT1: 57% [12/22 mice] versus 24% [4/19 mice], P<0.05) in model 2. Kidney and liver function did not change significantly. Minor liver inflammation was found in 56% of AAV9-sFLT1-treated model 1 mice. CONCLUSIONS: By applying a regulated mechanism to restrict sFLT1 expression to bAVM, AAV9-sFLT1 can potentially be developed into a safer therapy to reduce the bAVM severity.

CRISPR/Cas9-Mediated Genome Editing as a Therapeutic Approach for Leber Congenital Amaurosis 10.

As the most common subtype of Leber congenital amaurosis (LCA), LCA10 is a severe retinal dystrophy caused by mutations in the CEP290 gene. The most frequent mutation found in patients with LCA10 is a deep intronic mutation in CEP290 that generates a cryptic splice donor site. The large size of the CEP290 gene prevents its use in adeno-associated virus (AAV)-mediated gene augmentation therapy. Here, we show that targeted genomic deletion using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system represents a promising therapeutic approach for the treatment of patients with LCA10 bearing the CEP290 splice mutation. We generated a cellular model of LCA10 by introducing the CEP290 splice mutation into 293FT cells and we showed that guide RNA pairs coupled with SpCas9 were highly efficient at removing the intronic splice mutation and restoring the expression of wild-type CEP290. In addition, we demonstrated that a dual AAV system could effectively delete an intronic fragment of the Cep290 gene in the mouse retina. To minimize the immune response to prolonged expression of SpCas9, we developed a self-limiting CRISPR/Cas9 system that minimizes the duration of SpCas9 expression. These results support further studies to determine the therapeutic potential of CRISPR/Cas9-based strategies for the treatment of patients with LCA10.

Analytical Ultracentrifugation as an Approach to Characterize Recombinant Adeno-Associated Viral Vectors.

Recombinant adeno-associated viral (rAAV) vectors represent a novel class of biopharmaceutical drugs. The production of clinical-grade rAAV vectors for gene therapy would benefit from analytical methods that are able to monitor drug product quality with regard to homogeneity, purity, and manufacturing consistency. Here, we demonstrate the novel application of analytical ultracentrifugation (AUC) to characterize the homogeneity of preparations of rAAV vectors. We show that a single sedimentation velocity run of rAAV vectors detected and quantified a number of different viral species, such as vectors harboring an intact genome, lacking a vector genome (empty particles), and containing fragmented or incomplete vector genomes. This information is obtained by direct boundary modeling of the AUC data generated from refractometric or UV detection systems using the computer program SEDFIT. Using AUC, we show that multiple parameters contributed to vector quality, including the AAV genome form (i.e., self-complementary vs. single-stranded), vector genome size, and the production and purification methods. Hence, AUC is a critical tool for identifying optimal production and purification processes and for monitoring the physical attributes of rAAV vectors to ensure their quality.

Gene Therapies for Neovascular Age-Related Macular Degeneration.

Pathological neovascularization is a key component of the neovascular form (also known as the wet form) of age-related macular degeneration (AMD) and proliferative diabetic retinopathy. Several preclinical studies have shown that antiangiogenesis strategies are effective for treating neovascular AMD in animal models. Vascular endothelial growth factor (VEGF) is one of the main inducers of ocular neovascularization, and several clinical trials have shown the benefits of neutralizing VEGF in patients with neovascular AMD or diabetic macular edema. In this review, we summarize several preclinical and early-stage clinical trials with intraocular gene therapies, which have the potential to reduce or eliminate the repeated intravitreal injections that are currently required for the treatment of neovascular AMD.

Interleukin-17 retinotoxicity is prevented by gene transfer of a soluble interleukin-17 receptor acting as a cytokine blocker: implications for age-related macular degeneration.

Age-related macular degeneration (AMD) is a common yet complex retinal degeneration that causes irreversible central blindness in the elderly. Pathology is widely believed to follow loss of retinal pigment epithelium (RPE) and photoreceptor degeneration. Here we report aberrant expression of interleukin-17A (IL17A) and the receptor IL17RC in the macula of AMD patients. In vitro, IL17A induces RPE cell death characterized by the accumulation of cytoplasmic lipids and autophagosomes with subsequent activation of pro-apoptotic Caspase-3 and Caspase-9. This pathology is reduced by siRNA knockdown of IL17RC. IL17-dependent retinal degeneration in a mouse model of focal retinal degeneration can be prevented by gene therapy with adeno-associated virus vector encoding soluble IL17 receptor. This intervention rescues RPE and photoreceptors in a MAPK-dependent process. The IL17 pathway plays a key role in RPE and photoreceptor degeneration and could hold therapeutic potential in AMD.

Translational fidelity of intrathecal delivery of self-complementary AAV9-survival motor neuron 1 for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by mutations in survival motor neuron 1 (SMN1). Previously, we showed that central nervous system (CNS) delivery of an adeno-associated viral (AAV) vector encoding SMN1 produced significant improvements in survival in a mouse model of SMA. Here, we performed a dose-response study in SMA mice to determine the levels of SMN in the spinal cord necessary for efficacy, and measured the efficiency of motor neuron transduction in the spinal cord after intrathecal delivery in pigs and nonhuman primates (NHPs). CNS injections of 5e10, 1e10, and 1e9 genome copies (gc) of self-complementary AAV9 (scAAV9)-hSMN1 into SMA mice extended their survival from 17 to 153, 70, and 18 days, respectively. Spinal cords treated with 5e10, 1e10, and 1e9 gc showed that 70-170%, 30-100%, and 10-20% of wild-type levels of SMN were attained, respectively. Furthermore, detectable SMN expression in a minimum of 30% motor neurons correlated with efficacy. A comprehensive analysis showed that intrathecal delivery of 2.5e13 gc of scAAV9-GFP transduced 25-75% of the spinal cord motor neurons in NHPs. Thus, the extent of gene expression in motor neurons necessary to confer efficacy in SMA mice could be obtained in large-animal models, justifying the continual development of gene therapy for SMA.

Aurintricarboxylic acid increases yield of HSV-1 vectors.

Production of large quantities of viral vectors is crucial for the success of gene therapy in the clinic. There is a need for higher titers of herpes simplex virus-1 (HSV-1) vectors both for therapeutic use as well as in the manufacturing of clinical grade adeno-associated virus (AAV) vectors. HSV-1 yield increased when primary human fibroblasts were treated with anti-inflammatory drugs like dexamethasone or valproic acid. In our search for compounds that would increase HSV-1 yield, we investigated another anti-inflammatory compound, aurintricarboxylic acid (ATA). Although ATA has been previously shown to have antiviral effects, we find that low (micromolar) concentrations of ATA increased HSV-1 vector production yields. Our results showing the use of ATA to increase HSV-1 titers have important implications for the production of certain HSV-1 vectors as well as recombinant AAV vectors.

Prevalence of neutralizing factors against adeno-associated virus types 2 in age-related macular degeneration and polypoidal choroidal vasculopathy.

Adeno-associated virus type 2 (AAV2) mediated gene therapy providing a potential treatment in the eye. However, immune responses can limit virally mediated gene transfer and therapy. To assess preexisting AAV2 neutralizing factors (NF) titers in peripheral blood and the vitreous in patients with age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). 130 subjects were enrolled: 50 with neovascular AMD, 30 with PCV, and 50 controls. The serum and the vitreous were obtained for AAV2 NF assay. We found AAV2 NF are present in all of AMD, PCV patients and controls we tested. There were no significant differences in prevalence of NAb in serum between AMD, PCV and controls (P=0.999). There was no correlation between NF in serum and in vitreous (P>0.05), and NF in vitreous was significantly less than in serum. Our results for the first time showed in Chinese population, NF against AAV2 was present in serum of all the patients with AMD or PCV and controls, and there were no significant differences among these groups. Therefore, it demonstrated there were no correlations between AAV2 NF titer and these diseases. We found NF in vitreous was considerably less than in serum in all groups. We also found no direct correlation between NF in vitreous and in serum suggesting serum antibody levels may not be used to predict their counterparts in the vitreous. Our results will provide crucial information for future clinical studies in the development of new therapies based on AAV2 mediated gene delivery in the eye.

AAV5-mediated sFLT01 gene therapy arrests retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) mice.

To test the effects of adeno-associated virus encoding sFLT01 (AAV5.sFLT01) on the retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) mice, a model for age-related macular degeneration (AMD), AAV5.sFLT01 was injected into the subretinal space of the right eyes and the left eyes served as controls. Histology found no retinal toxicity due to the treatment after 3 months. The treated eyes showed lesion arrest compared with lesion progression in the left eyes by fundus monitoring monthly and histological evaluation 3 months after treatment. Retinal ultrastructure showed fewer lipofuscin and better preserved photoreceptors after the treatment. A2E, a major component of lipofuscin, was lower in the treated eyes than in the control eyes. Molecular analysis showed that AAV5.sFLT01 lowered retinal extracellular signal-regulated kinase (ERK) phosphorylation and inducible nitric oxide synthetase expression, which suggested the involvement of reactive nitrogen species in the retinal lesions of Ccl2(-/-)/Cx3cr1(-/-). We concluded that local delivery of AAV5.sFLT01 can stabilize retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) mice. The findings provide further support for the potential beneficial effects of sFLT01 gene therapy for age-related macular degeneration.