A highly efficient transgene knock-in technology in clinically relevant cell types.

Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.

The Impact of Inherited Retinal Diseases in the United States of America (US) and Canada from a Cost-of-Illness Perspective.

OBJECTIVE: To estimate the annual cost of inherited retinal diseases (IRDs) in the United States of America (US) and Canada from a societal perspective - including costs to the health system, individual and family productivity costs, lost wellbeing and other societal economic costs - by setting and payer. Findings will inform the need for policy action to mitigate the impact of IRDs. METHODS: The costs of IRDs were estimated using a cost-of-illness methodology, based on the prevalence of IRDs in each country. Intangible costs of reduced wellbeing were also estimated using disability-adjusted life years which were then converted to monetary values using the value of a statistical life. RESULTS: Using base prevalence rates, total costs attributable to IRDs in the US were estimated to range between US$13,414.0 and US$31,797.4 million in 2019, comprising both economic costs (between US$4,982 and US$11,753.9 million; 37% of total costs) and wellbeing costs (between US$8,431.7 and US$20,043.6 million; 63%). Total costs attributable to IRDs in Canada were estimated to range between CAN$1637.8 and CAN$6687.5 million in 2019, comprising both economic costs (between CAN$566.6 and CAN$2,305.7 million; 34%) and wellbeing costs (between CAN$1,071.4 and CAN$4,381.9 million; 66% of total costs). CONCLUSION: The impact of IRDs in the US and Canada is substantial when considering both economic costs and reduced wellbeing. The wellbeing costs due to IRDs in the US and Canada are considerable, accounting for over 60% of total costs. Vision loss from IRDs often manifests in childhood, meaning some people live with vision impairment and blindness for their whole lives. Further research into current and emerging cost-effective therapies and interventions is required given the substantial economic burden faced by those living with vision loss.

Dose Range Finding Studies with Two RPGR Transgenes in a Canine Model of X-Linked Retinitis Pigmentosa Treated with Subretinal Gene Therapy.

Recombinant adeno-associated viral (rAAV) vector-mediated gene therapy is being developed to treat X-linked retinitis pigmentosa (XLRP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. In preparation for a clinical gene therapy trial, we conducted dose range finding (DRF) studies with an AAV2 capsid with three surface tyrosine residues changed to phenylalanine (AAV2tYF) vector administered by subretinal injection in a naturally occurring RPGR-mutant canine model (XLPRA2) to compare two different human RPGR (hRPGR) transgenes and to establish a reasonable starting dose for a clinical trial. Different dose levels of two candidate vectors (0.15 mL at 1.2 × 1010-3.0 × 1012 vg/mL of rAAV2tYF-GRK1-hRPGRco or 4 × 1010-3.0 × 1012 vg/mL of rAAV2tYF-GRK1-hRPGRstb), 6.0 × 1011 vg/mL rAAV5-GRK1-hRPGRco reference vector or Vehicle were subretinally administered, and the dogs were followed for 8 weeks postdose. Ophthalmic examinations, analyses of retinal structure by in vivo imaging using confocal scanning laser ophthalmoscopy (cSLO)/optical coherence tomography (OCT) in the Lower (4.0 × 1010 vg/mL) and Lowest (1.2 × 1010 vg/mL) Doses, immunological responses by cell based assays or enzyme-linked immunosorbent assay, RPGR transgene expression, and reversal of opsin mislocalization by immunohistochemistry were performed. No sustained signs of ocular discomfort or ophthalmic complications were noted in any of the injected eyes except some in the High Dose group (3.0 × 1012 vg/mL), which showed signs of retinal detachment and inflammation. A change in fundus reflectivity suggestive of a rescue effect was seen in the High, Mid (6.0 × 1011 vg/mL), and Low (1.2 × 1011 vg/mL) Dose groups. cSLO/OCT demonstrated qualitative and quantitative evidence of rescue effect in eyes treated with the Lower Dose. Anti-hRPGR antibodies were absent, but neutralizing antibody titers against AAV2 were detected in all animals dosed with rAAV2tYF in an apparent dose-related pattern. RPGR expression was stronger for rAAV2tYF-GRK1-hRPGRco compared to rAAV2tYF-GRK1-hRPGRstb at all dose levels. Subretinal administration of rAAV2tYF-GRK1-hRPGRco and rAAV2tYF-GRK1-hRPGRstb both corrected rod and cone opsin mislocalization, two early markers of disease in the XLPRA2 canine model of RPGR-XLRP. These results support the selection and use of rAAV2tYF-GRK1-hRPGRco (AGTC-501) and guided the initial doses in clinical studies in patients with XLRP caused by RPGR mutations.

Toxicity and Efficacy Evaluation of an Adeno-Associated Virus Vector Expressing Codon-Optimized RPGR Delivered by Subretinal Injection in a Canine Model of X-linked Retinitis Pigmentosa.

Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector AGTC-501, also designated rAAV2tYF-GRK1-hRPGRco, to treat X-linked retinitis pigmentosa (XLRP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The vector contains a codon-optimized human RPGR cDNA (hRPGRco) driven by a photoreceptor-specific promoter (G protein-coupled receptor kinase 1 [GRK1]), and is packaged in an AAV2 capsid variant with three surface tyrosine residues changed to phenylalanine (AAV2tYF). We conducted a toxicity and efficacy study of this vector administered by subretinal injection in the naturally occurring RPGR mutant (X-linked progressive retinal atrophy 2 [XLPRA2]) dog model. Sixteen RPGR mutant dogs divided into four groups of three to five animals each received either a subretinal injection of 0.07 mL of AGTC-501 at low (1.2 × 1011 vector genome [vg]/mL), mid (6 × 1011 vg/mL), or high dose (3 × 1012 vg/mL), or of vehicle control in the right eye at early-stage disease. The left eye remained untreated. Subretinal injections were well tolerated and were not associated with systemic toxicity. Electroretinography, in vivo retinal imaging, and histological analysis showed rescue of photoreceptor function and structure in the absence of ocular toxicity in the low- and mid-dose treatment groups when compared with the vehicle-treated group. The high-dose group showed evidence of both photoreceptor rescue and posterior segment toxicity. These results support the use of AGTC-501 in clinical studies with patients affected with XLRP caused by RPGR mutations and define the no-observed-adverse-effect level at 6 × 1011 vg/mL.

Ocular Inflammatory Response to Intravitreal Injection of Adeno-Associated Virus Vector: Relative Contribution of Genome and Capsid.

Both subretinal dosing and intravitreal (IVT) dosing of adeno-associated virus (AAV) in higher species induce mild and transient inflammatory responses that increase with dose. Foreign protein and foreign DNA are known inducers of inflammation, which is also true in the immune-privileged ocular environment. We explored which component(s) of AAV vectors, viral capsid, or viral DNA drive inflammatory responses. Recombinant AAV with three tyrosine to phenylalanine substitutions in the capsid of AAV serotype 2 (rAAV2tYF), and with a generic ubiquitous promoter (cytomegalovirus [CMV]) controlling the expression of humanized green fluorescent protein (hGFP), was processed to enrich for AAV capsids containing genome (full capsids), capsids without genome (empty capsids), and residual material. Nonhuman primate eyes were injected by IVT in both eyes. During in-life, ocular inflammation and development of neutralizing antibodies (NAb) were measured. Following termination, lymph node immunophenotyping was performed, vitreous was processed for cytokine and RNAseq analyses, and ocular sections were assessed for transgene expression (by in situ hybridization) and histopathology. IVT dosing of AAV vectors transiently raised cellular inflammation in the aqueous and induced a more sustained inflammation in the vitreous. Lowering the total capsid dose by removing empty AAV capsids reduced inflammation and improved viral transduction. IVT dosing of AAV induced systemic NAb to AAV irrespective of the vector preparation. Similarly, lymph node immunophenotyping revealed identical profiles irrespective of viral preparation used for dosing. Immune cells in the vitreous were identified based on RNAseq analysis. Three months postdose, cytokine levels were low, indicative of minimal levels of inflammation in agreement with histopathological assessment of the retina.

Toxicology and Pharmacology of an AAV Vector Expressing Codon-Optimized RPGR in RPGR-Deficient Rd9 Mice.

Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector AGTC-501, also designated AAV2tYF-GRK1-RPGRco, to treat retinitis pigmentosa (RP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The vector contains a codon-optimized human RPGR cDNA (RPGRco) driven by a photoreceptor-specific promoter (G protein-coupled receptor kinase 1, GRK1) and is packaged in an AAV2 capsid with three surface tyrosine residues changed to phenylalanine (AAV2tYF). We conducted a safety and potency study of this vector administered by subretinal a injection in the naturally occurring RPGR-deficient Rd9 mouse model. Sixty Rd9 mice (20 per group) received a subretinal injection in the right eye of vehicle (control) or AAV2tYF-GRK1-RPGRco at one of two dose levels (4 × 108 or 4 × 109 vg/eye) and were followed for 12 weeks after injection. Vector injections were well tolerated, with no systemic toxicity. There was a trend towards reduced electroretinography b-wave amplitudes in the high vector dose group that was not statistically significant. There were no clinically important changes in hematology or clinical chemistry parameters and no vector-related ocular changes in life or by histological examination. Dose-dependent RPGR protein expression, mainly in the inner segment of photoreceptors and the adjacent connecting cilium region, was observed in all vector-treated eyes examined. Sequence integrity of the codon-optimized RPGR was confirmed by sequencing of PCR-amplified DNA, or cDNA reverse transcribed from total RNA extracted from vector-treated retinal tissues, and by sequencing of RPGR protein obtained from transfected HEK 293 cells. These results support the use of rAAV2tYF-GRK1-RPGRco in clinical studies in patients with XLRP caused by RPGR mutations.

Optimization of Retinal Gene Therapy for X-Linked Retinitis Pigmentosa Due to RPGR Mutations.

X-linked retinitis pigmentosa (XLRP) caused by mutations in the RPGR gene is an early onset and severe cause of blindness. Successful proof-of-concept studies in a canine model have recently shown that development of a corrective gene therapy for RPGR-XLRP may now be an attainable goal. In preparation for a future clinical trial, we have here optimized the therapeutic AAV vector construct by showing that GRK1 (rather than IRBP) is a more efficient promoter for targeting gene expression to both rods and cones in non-human primates. Two transgenes were used in RPGR mutant (XLPRA2) dogs under the control of the GRK1 promoter. First was the previously developed stabilized human RPGR (hRPGRstb). Second was a new full-length stabilized and codon-optimized human RPGR (hRPGRco). Long-term (>2 years) studies with an AAV2/5 vector carrying hRPGRstb under control of the GRK1 promoter showed rescue of rods and cones from degeneration and retention of vision. Shorter term (3 months) studies demonstrated comparable preservation of photoreceptors in canine eyes treated with an AAV2/5 vector carrying either transgene under the control of the GRK1 promoter. These results provide the critical molecular components (GRK1 promoter, hRPGRco transgene) to now construct a therapeutic viral vector optimized for RPGR-XLRP patients.

Safety and Efficacy Evaluation of rAAV2tYF-PR1.7-hCNGA3 Vector Delivered by Subretinal Injection in CNGA3 Mutant Achromatopsia Sheep.

Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector expressing the human CNGA3 gene designated AGTC-402 (rAAV2tYF-PR1.7-hCNGA3) for the treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. The results are herein reported of a study evaluating safety and efficacy of AGTC-402 in CNGA3-deficient sheep. Thirteen day-blind sheep divided into three groups of four or five animals each received a subretinal injection of an AAV vector expressing a CNGA3 gene in a volume of 500 µL in the right eye. Two groups (n = 9) received either a lower or higher dose of the AGTC-402 vector, and one efficacy control group (n = 4) received a vector similar in design to one previously shown to rescue cone photoreceptor responses in the day-blind sheep model (rAAV5-PR2.1-hCNGA3). The left eye of each animal received a subretinal injection of 500 µL of vehicle (n = 4) or was untreated (n = 9). Subretinal injections were generally well tolerated and not associated with systemic toxicity. Most animals had mild to moderate conjunctival hyperemia, chemosis, and subconjunctival hemorrhage immediately after surgery that generally resolved by postoperative day 7. Two animals treated with the higher dose of AGTC-402 and three of the efficacy control group animals had microscopic findings of outer retinal atrophy with or without inflammatory cells in the retina and choroid that were procedural and/or test-article related. All vector-treated eyes showed improved cone-mediated electroretinography responses with no change in rod-mediated electroretinography responses. Behavioral maze testing under photopic conditions showed significantly improved navigation times and reduced numbers of obstacle collisions in all vector-treated eyes compared to their contralateral control eyes or pre-dose results in the treated eyes. These results support the use of AGTC-402 in clinical studies in patients with achromatopsia caused by CNGA3 mutations, with careful evaluation for possible inflammatory and/or toxic effects.

Potent benzoazepinone γ-secretase modulators with improved bioavailability.

The triazolyl amide γ-secretase modulators are potent alternatives to the cinnamyl amides that have entered the clinic for the treatment of Alzheimer's disease. Herein we build on the lead benzoazepinones described in our prior communication with imidazomethoxyarene moiety alternatives that offer opportunities to fine tune physical properties as well as address hERG binding and PK. Both half-life and bioavailability were significantly improved, especially in dog, with robust brain Aß42 lowering maintained in both transgenic mouse and rat.

Discovery of novel triazolobenzazepinones as γ-secretase modulators with central Aß42 lowering in rodents and rhesus monkeys.

Synthesis and SAR studies of novel triazolobenzazepinones as gamma secretase modulators (GSMs) are presented in this communication. Starting from our azepinone leads, optimization studies toward improving central lowering of Aß42 led to the discovery of novel benzo-fused azepinones. Several benzazepinones were profiled in vivo and found to lower brain Aß42 levels in Sprague Dawley rats and transgenic APP-YAC mice in a dose-dependent manner after a single oral dose. Compound 34 was further progressed into a pilot study in our cisterna-magna-ported rhesus monkey model, where we observed robust lowering of CSF Aß42 levels.

Pathway-based analysis of genome-wide siRNA screens reveals the regulatory landscape of APP processing.

The progressive aggregation of Amyloid-ß (Aß) in the brain is a major trait of Alzheimer's Disease (AD). Aß is produced as a result of proteolytic processing of the ß-amyloid precursor protein (APP). Processing of APP is mediated by multiple enzymes, resulting in the production of distinct peptide products: the non-amyloidogenic peptide sAPPα and the amyloidogenic peptides sAPPß, Aß40, and Aß42. Using a pathway-based approach, we analyzed a large-scale siRNA screen that measured the production of different APP proteolytic products. Our analysis identified many of the biological processes/pathways that are known to regulate APP processing and have been implicated in AD pathogenesis, as well as revealing novel regulatory mechanisms. Furthermore, we also demonstrate that some of these processes differentially regulate APP processing, with some mechanisms favouring production of certain peptide species over others. For example, synaptic transmission having a bias towards regulating Aß40 production over Aß42 as well as processes involved in insulin and pancreatic biology having a bias for sAPPß production over sAPPα. In addition, some of the pathways identified as regulators of APP processing contain genes (CLU, BIN1, CR1, PICALM, TREM2, SORL1, MEF2C, DSG2, EPH1A) recently implicated with AD through genome wide association studies (GWAS) and associated meta-analysis. In addition, we provide supporting evidence and a deeper mechanistic understanding of the role of diabetes in AD. The identification of these processes/pathways, their differential impact on APP processing, and their relationships to each other, provide a comprehensive systems biology view of the "regulatory landscape" of APP.

Increased O-GlcNAcylation reduces pathological tau without affecting its normal phosphorylation in a mouse model of tauopathy.

Neurofibrillary tangles (NFT), mainly consisting of fibrillar aggregates of hyperphosphorylated tau, are a defining pathological feature of Alzheimer's Disease and other tauopathies. Progressive accumulation of tau into NFT is considered to be a toxic cellular event causing neurodegeneration. Tau is subject to O-linked N-acetylglucosamine (O-GlcNAc) modification and O-GlcNAcylation of tau has been suggested to regulate tau phosphorylation. We tested if an increase in tau O-GlcNAcylation affected tau phosphorylation and aggregation in the rTg4510 tau transgenic mouse model. Acute treatment of rTg4510 mice with an O-GlcNAcase inhibitor transiently reduced tau phosphorylation at epitopes implicated in tau pathology. More importantly, long-term inhibitor treatment strongly increased tau O-GlcNAcylation, reduced the number of dystrophic neurons, and protected against the formation of pathological tau species without altering the phosphorylation of non-pathological tau. This indicates that O-GlcNAcylation prevents the aggregation of tau in a manner that does not affect its normal phosphorylation state. Collectively, our results support O-GlcNAcase inhibition as a potential therapeutic strategy for the treatment of Alzheimer's Disease and other tauopathies.

Triazoloamides as potent γ-secretase modulators with reduced hERG liability.

Synthesis and SAR studies of novel aryl triazoles as gamma secretase modulators (GSMs) are presented in this communication. Starting from our aryl triazole leads, optimization studies were continued and the series progressed towards novel amides and lactams. Triazole 57 was identified as the most potent analog in this series, displaying single-digit nanomolar Aß42 IC(50) in cell-based assays and reduced affinity for the hERG channel.

Triazoles as γ-secretase modulators.

Synthesis, SAR, and evaluation of aryl triazoles as novel gamma secretase modulators (GSMs) are presented in this communication. Starting from the literature and in-house leads, we evaluated a range of five-membered heterocycles as replacements for olefins commonly found in non-acid GSMs. 1,2,3-C-aryl-triazoles were identified as suitable replacements which exhibited good modulation of γ-secretase activity, excellent pharmacokinetics and good central lowering of Aß42 in Sprague-Dawley rats.

Quinazolinones as γ-secretase modulators.

Synthesis, SAR and evaluation of styrenyl quinazolinones as novel gamma secretase modulators are presented in this communication. Starting from literature and in-house leads we evaluated a range of quinazolinones which showed good modulation of γ-secretase activity.

Acute gamma-secretase inhibition of nonhuman primate CNS shifts amyloid precursor protein (APP) metabolism from amyloid-beta production to alternative APP fragments without amyloid-beta rebound.

The accumulation of amyloid beta (Abeta) in Alzheimer's disease is caused by an imbalance of production and clearance, which leads to increased soluble Abeta species and extracellular plaque formation in the brain. Multiple Abeta-lowering therapies are currently in development: an important goal is to characterize the molecular mechanisms of action and effects on physiological processing of Abeta, as well as other amyloid precursor protein (APP) metabolites, in models which approximate human Abeta physiology. To this end, we report the translation of the human in vivo stable-isotope-labeling kinetics (SILK) method to a rhesus monkey cisterna magna ported (CMP) nonhuman primate model, and use the model to test the mechanisms of action of a gamma-secretase inhibitor (GSI). A major concern of inhibiting the enzymes which produce Abeta (beta- and gamma-secretase) is that precursors of Abeta may accumulate and cause a rapid increase in Abeta production when enzyme inhibition discontinues. In this study, the GSI MK-0752 was administered to conscious CMP rhesus monkeys in conjunction with in vivo stable-isotope-labeling, and dose-dependently reduced newly generated CNS Abeta. In contrast to systemic Abeta metabolism, CNS Abeta production was not increased after the GSI was cleared. These results indicate that most of the CNS APP was metabolized to products other than Abeta, including C-terminal truncated forms of Abeta: 1-14, 1-15 and 1-16; this demonstrates an alternative degradation pathway for CNS amyloid precursor protein during gamma-secretase inhibition.

Purine derivatives as potent gamma-secretase modulators.

The development of a novel series of purines as gamma-secretase modulators for potential use in the treatment of Alzheimer's disease is disclosed herein. Optimization of a previously disclosed pyrimidine series afforded a series of potent purine-based gamma-secretase modulators with 300- to 2000-fold in vitro selectivity over inhibition of Notch cleavage and that selectively reduces Alphabeta42 in an APP-YAC transgenic mouse model.

Oral treatment with a gamma-secretase inhibitor improves long-term potentiation in a mouse model of Alzheimer's disease.

The beta-amyloid peptide (Abeta) is thought to play a critical role in the pathophysiology of Alzheimer's disease (AD). To study the effects of Abeta on the brain, transgenic mouse models have been developed that express high levels of Abeta. These mice show some features of AD, including amyloid plaques and mild cognitive impairment, but not others such as progressive neurodegeneration. We investigated the age-dependent effects of Abeta on synaptic physiology in Tg2576 mice that express human Abeta. We report that both basal synaptic activity and long-term potentiation (LTP), as measured in the CA1 region of the hippocampus, were compromised by 7 months of age before plaque deposition. Despite a persistent increase in Abeta levels with age, LTP recovered in 14-month-old mice, with no further loss of basal activity compared with activity measured in 7-month-old mice. Previous work has shown that inhibitors of gamma-secretase, an enzyme critical for Abeta synthesis, can significantly reduce Abeta production and plaque formation in Tg2576 mice. Our data demonstrate that 7-month-old Tg2576 mice treated with an orally available gamma-secretase inhibitor showed a significant improvement in synaptic function and plasticity within days, and the effect was correlated with the extent and duration of Abeta reduction. These results indicate that recovery from Abeta-mediated synaptotoxicity can occur rapidly with Abeta-lowering therapies. These findings highlight some of the strengths and limitations of using Abeta-overexpressing mouse models for Alzheimer's drug discovery.

Piperazinyl pyrimidine derivatives as potent gamma-secretase modulators.

The development of a novel series of piperazinyl pyrimidines as gamma-secretase modulators for potential use in the treatment of Alzheimer's disease is disclosed herein. Optimization of a screening hit provided a series of potent gamma-secretase modulators with >180-fold in vitro selectivity over inhibition of Notch cleavage.