Potent and sustained huntingtin lowering via AAV5 encoding miRNA preserves striatal volume and cognitive function in a humanized mouse model of Huntington disease.

Huntington disease (HD) is a fatal neurodegenerative disease caused by a pathogenic expansion of a CAG repeat in the huntingtin (HTT) gene. There are no disease-modifying therapies for HD. Artificial microRNAs targeting HTT transcripts for degradation have shown preclinical promise and will soon enter human clinical trials. Here, we examine the tolerability and efficacy of non-selective HTT lowering with an AAV5 encoded miRNA targeting human HTT (AAV5-miHTT) in the humanized Hu128/21 mouse model of HD. We show that intrastriatal administration of AAV5-miHTT results in potent and sustained HTT suppression for at least 7 months post-injection. Importantly, non-selective suppression of huntingtin was generally tolerated, however high dose AAV5-miHTT did induce astrogliosis. We observed an improvement of select behavioural and modest neuropathological HD-like phenotypes in Hu128/21 mice, suggesting a potential therapeutic benefit of miRNA-mediated non-selective HTT lowering. Finally, we also observed that potent reduction of wild type HTT (wtHTT) in Hu21 control mice was tolerated up to 7 months post-injection but may induce impairment of motor coordination and striatal atrophy. Taken together, our data suggests that in the context of HD, the therapeutic benefits of mHTT reduction may outweigh the potentially detrimental effects of wtHTT loss following non-selective HTT lowering.

AAV5-miHTT Gene Therapy Demonstrates Broad Distribution and Strong Human Mutant Huntingtin Lowering in a Huntington's Disease Minipig Model.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Previously, we showed strong huntingtin reduction and prevention of neuronal dysfunction in HD rodents using an engineered microRNA targeting human huntingtin, delivered via adeno-associated virus (AAV) serotype 5 vector with a transgene encoding an engineered miRNA against HTT mRNA (AAV5-miHTT). One of the challenges of rodents as a model of neurodegenerative diseases is their relatively small brain, making successful translation to the HD patient difficult. This is particularly relevant for gene therapy approaches, where distribution achieved upon local administration into the parenchyma is likely dependent on brain size and structure. Here, we aimed to demonstrate the translation of huntingtin-lowering gene therapy to a large-animal brain. We investigated the feasibility, efficacy, and tolerability of one-time intracranial administration of AAV5-miHTT in the transgenic HD (tgHD) minipig model. We detected widespread dose-dependent distribution of AAV5-miHTT throughout the tgHD minipig brain that correlated with the engineered microRNA expression. Both human mutant huntingtin mRNA and protein were significantly reduced in all brain regions transduced by AAV5-miHTT. The combination of widespread vector distribution and extensive huntingtin lowering observed with AAV5-miHTT supports the translation of a huntingtin-lowering gene therapy for HD from preclinical studies into the clinic.

Cholesterol ester transfer protein inhibition by TA-8995 in patients with mild dyslipidaemia (TULIP): a randomised, double-blind, placebo-controlled phase 2 trial.

BACKGROUND: Dyslipidaemia remains a significant risk factor for cardiovascular disease and additional lipid-modifying treatments are warranted to further decrease the cardiovascular disease burden. We assessed the safety, tolerability and efficacy of a novel cholesterol esterase transfer protein (CETP) inhibitor TA-8995 in patients with mild dyslipidaemia. METHODS: In this randomised, double-blind, placebo-controlled, parallel-group phase 2 trial, we recruited patients (aged 18-75 years) from 17 sites (hospitals and independent clinical research organisations) in the Netherlands and Denmark with fasting LDL cholesterol levels between 2·5 mmol/L and 4·5 mmol/L, HDL cholesterol levels between 0·8 and 1·8 mmol/L and triglyceride levels below 4·5 mmol/L after washout of lipid-lowering treatments. Patients were randomly allocated (1:1) by a computer-generated randomisation schedule to receive one of the following nine treatments: a once a day dose of 1 mg, 2·5 mg, 5 mg, or 10 mg TA-8995 or matching placebo; 10 mg TA-8995 plus 20 mg atorvastatin; 10 mg TA-8995 plus 10 mg rosuvastatin or 20 mg atorvastatin or 10 mg rosuvastatin alone. We overencapsulated statins to achieve masking. The primary outcome was percentage change in LDL cholesterol and HDL cholesterol from baseline at week 12, analysed by intention to treat. This study is registered with ClinicalTrials.gov, number NCT01970215. FINDINGS: Between Aug 15, 2013, and Jan 10, 2014, 364 patients were enrolled. At week 12, LDL cholesterol levels were reduced by 27·4% in patients assigned to the 1 mg dose, 32·7% in patients given the 2·5 mg dose, 45·3% in those given the 5 mg dose, and 45·3% in those given the 10 mg dose (p<0·0001). LDL cholesterol levels were reduced by 68·2% in patients given 10 mg TA-8995 plus atorvastatin, and by 63·3% in patients given rosuvastatin plus 10 mg TA-8995 (p<0·0001). A daily dose of 1 mg TA-8995 increased HDL cholesterol levels by 75·8%, 2·5 mg by 124·3%, 5 mg by 157·1%, and 10 mg dose by 179·0% (p<0·0001). In patients receiving 10 mg TA-8995 and 20 mg atorvastatin HDL cholesterol levels increased by 152·1% and in patients receiving 10 mg TA-8995 and 10 mg rosuvastatin by 157·5%. We recorded no serious adverse events or signs of liver or muscle toxic effects. INTERPRETATION: TA-8995, a novel CETP inhibitor, is well tolerated and has beneficial effects on lipids and apolipoproteins in patients with mild dyslipidaemia. A cardiovascular disease outcome trial is needed to translate these effects into a reduction of cardiovascular disease events. FUNDING: Dezima.

Mir-142-3p target sequences reduce transgene-directed immunogenicity following intramuscular adeno-associated virus 1 vector-mediated gene delivery.

BACKGROUND: Muscle represents an important tissue target for adeno-associated virus (AAV) vector-mediated gene transfer in muscular, metabolic or blood-related genetic disorders. However, several studies have demonstrated the appearance of immune responses against the transgene product after intramuscular AAV vector delivery that resulted in a limited efficacy of the treatment. Use of microRNAs that are specifically expressed in antigen-presenting cells (APCs) is a promising approach for avoiding those immune responses. Cellular mir-142-3p, which is APC-specific, is able to repress the translation of its target cellular transcripts by binding to a specific target sequences. METHODS: In the present study, we explored the potential of mir-142-3p specific target sequences with respect to reducing or abolishing immune responses directed against ovalbumin (OVA), a highly immunogenic protein, expressed as transgene and delivered by AAV1 vector administered intramuscularly. RESULTS: The occurrence of immune responses against OVA transgene following intramuscular delivery by AAV have been described previously and resulted in the loss of OVA protein expression. In the present study, we demonstrate that OVA protein expression was maintained when mir-142-3pT sequences were incorporated into the expression cassette. The sustained expression of OVA protein over time correlated with a reduced increase in anti-OVA antibody levels. Furthermore, no cellular infiltrates were observed in the muscle tissue when AAV1 vectors containing four or eight repeats of mir-142-3p target sequences after the OVA sequence were used. CONCLUSIONS: The rising humoral and cellular immune responses against OVA protein after intramuscular delivery can be efficiently reduced by the use of mir-142-3p target sequences.

Adenosine triphosphate-binding cassette transporter genes up-regulation in untreated hepatocellular carcinoma is mediated by cellular microRNAs.

UNLABELLED: Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are drug efflux pumps responsible for the multidrug resistance phenotype causing hepatocellular carcinoma (HCC) treatment failure. Here we studied the expression of 15 ABC transporters relevant for multidrug resistance in 19 paired HCC patient samples (16 untreated, 3 treated by chemotherapeutics). Twelve ABC transporters showed up-regulation in HCC compared with adjacent healthy liver. These include ABCA2, ABCB1, ABCB6, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC10, ABCC11, ABCC12, and ABCE1. The expression profile and function of some of these transporters have not been associated with HCC thus far. Because cellular microRNAs (miRNAs) are involved in posttranscriptional gene silencing, we hypothesized that regulation of ABC expression in HCC might be mediated by miRNAs. To study this, miRNAs were profiled and dysregulation of 90 miRNAs was shown in HCC compared with healthy liver, including up-regulation of 11 and down-regulation of 79. miRNA target sites in ABC genes were bioinformatically predicted and experimentally verified in vitro using luciferase reporter assays. In total, 13 cellular miRNAs were confirmed that target ABCA1, ABCC1, ABCC5, ABCC10, and ABCE1 genes and mediate changes in gene expression. Correlation analysis between ABC and miRNA expression in individual patients revealed an inverse relationship, providing an indication for miRNA regulation of ABC genes in HCC. CONCLUSION: Up-regulation of ABC transporters in HCC occurs prior to chemotherapeutic treatment and is associated with miRNA down-regulation. Up-regulation of five ABC genes appears to be mediated by 13 cellular miRNAs in HCC patient samples. miRNA-based gene therapy may be a novel and promising way to affect the ABC profile and overcome clinical multidrug resistance.

Indian Hedgehog is an antagonist of Wnt signaling in colonic epithelial cell differentiation.

Wnt signaling defines the colonic epithelial progenitor cell phenotype, and mutations in the gene adenomatous polyposis coli (APC) that activate the Wnt pathway cause the familial adenomatous polyposis coli (FAP) syndrome and most sporadic colon cancers. The mechanisms that regulate the transition of epithelial precursor cells into their differentiated derivatives are poorly characterized. We report that Indian hedgehog (Ihh) is expressed by mature colonocytes and regulates their differentiation in vitro and in vivo. Hedgehog (Hh) signaling restricts the expression of Wnt targets to the base of the colonic crypt in vivo, and transfection of Ihh into colon cancer cells leads to a downregulation of both components of the nuclear TCF4-beta-catenin complex and abrogates endogenous Wnt signaling in vitro. In turn, expression of Ihh is downregulated in polyps of individuals with FAP and expression of doxycycline-inducible dominant negative TCF4 (dnTCF4) restores Ihh expression in APC mutant DLD-1 colon cancer cells. These data identify a new Wnt-Hh axis in colonic epithelial renewal.

Murine CD4⁺CD25⁻ cells activated in vitro with PMA/ionomycin and anti-CD3 acquire regulatory function and ameliorate experimental colitis in vivo.

BACKGROUND: Induced regulatory T (iTreg) lymphocytes show promise for application in the treatment of allergic, autoimmune and inflammatory disorders. iTreg cells demonstrate advantages over natural Treg (nTreg) cells in terms of increased number of starting population and greater potential to proliferate. Different activation methods to generate iTreg cells result in iTreg cells that are heterogeneous in phenotype and mechanisms of suppression. Therefore it is of interest to explore new techniques to generate iTreg cells and to determine their physiological relevance. METHODS: Using phorbol myristate acetate (PMA)/ionomycin and anti-CD3 activation of CD4⁺CD25⁻ cells we generated in vitro functional CD4⁺CD25⁻ iTreg (TregPMA) cells. Functionality of the generated TregPMA cells was tested in vivo in a mouse model of inflammatory bowel disease (IBD) - CD45RB transfer colitis model. RESULTS: TregPMA cells expressed regulatory markers and proved to ameliorate the disease phenotype in murine CD45RB transfer colitis model. The body weight loss and disease activity scores for TregPMA treated mice were reduced when compared to diseased control group. Histological assessment of colon sections confirmed amelioration of the disease phenotype. Additionally, cytokine analysis showed decreased levels of proinflammatory colonic and plasma IL-6, colonic IL-1 ß and higher levels of colonic IL-17 when compared to diseased control group. CONCLUSIONS: This study identifies a new method to generate in vitro iTreg cells (TregPMA cells) which physiological efficacy has been demonstrated in vivo.

Induction of antigen-specific tolerance by oral administration of Lactococcus lactis delivered immunodominant DQ8-restricted gliadin peptide in sensitized nonobese diabetic Abo Dq8 transgenic mice.

Active delivery of recombinant autoantigens or allergens at the intestinal mucosa by genetically modified Lactococcus lactis (LL) provides a novel therapeutic approach for the induction of tolerance. Celiac disease is associated with either HLA-DQ2- or HLA-DQ8-restricted responses to specific antigenic epitopes of gliadin, and may be treated by induction of Ag-specific tolerance. We investigated whether oral administration of LL-delivered DQ8-specific gliadin epitope induces Ag-specific tolerance. LL was engineered to secrete a deamidated DQ8 gliadin epitope (LL-eDQ8d) and the induction of Ag-specific tolerance was studied in NOD AB degrees DQ8 transgenic mice. Tolerance was assessed by delayed-type hypersensitivity reaction, cytokine measurements, eDQ8d-specific proliferation, and regulatory T cell analysis. Oral administration of LL-eDQ8d induced suppression of local and systemic DQ8-restricted T cell responses in NOD AB degrees DQ8 transgenic mice. Treatment resulted in an Ag-specific decrease of the proliferative capacity of inguinal lymph node (ILN) cells and lamina propria cells. Production of IL-10 and TGF-beta and a significant induction of Foxp3(+) regulatory T cells were associated with the eDQ8d-specific suppression induced by LL-eDQ8d. These data provide support for the development of effective therapeutic approaches for gluten-sensitive disorders using orally administered Ag-secreting LL. Such treatments may be effective even in the setting of established hypersensitivity.

Secreted therapeutics: monitoring durability of microRNA-based gene therapies in the central nervous system.

The preclinical development of microRNA-based gene therapies for inherited neurodegenerative diseases is accompanied by translational challenges. Due to the inaccessibility of the brain to periodically evaluate therapy effects, accessible and reliable biomarkers indicative of dosing, durability and therapeutic efficacy in the central nervous system are very much needed. This is particularly important for viral vector-based gene therapies, in which a one-time administration results in long-term expression of active therapeutic molecules in the brain. Recently, extracellular vesicles have been identified as carriers of RNA species, including microRNAs, and proteins in all biological fluids, whilst becoming potential sources of biomarkers for diagnosis. In this study, we investigated the secretion and potential use of circulating miRNAs associated with extracellular vesicles as suitable sources to monitor the expression and durability of gene therapies in the brain. Neuronal cells derived from induced pluripotent stem cells were treated with adeno-associated viral vector serotype 5 carrying an engineered microRNA targeting huntingtin or ataxin3 gene sequences, the diseases-causing genes of Huntington disease and spinocerebellar ataxia type 3, respectively. After treatment, the secretion of mature engineered microRNA molecules was confirmed, with extracellular microRNA levels correlating with viral dose and cellular microRNA expression in neurons. We further investigated the detection of engineered microRNAs over time in the CSF of non-human primates after a single intrastriatal injection of adeno-associated viral vector serotype 5 carrying a huntingtin-targeting engineered microRNA. Quantifiable engineered microRNA levels enriched in extracellular vesicles were detected in the CSF up to 2 years after brain infusion. Altogether, these results confirm the long-term expression of adeno-associated viral vector serotype 5-delivered microRNAs and support the use of extracellular vesicle-associated microRNAs as novel translational pharmacokinetic markers in ongoing clinical trials of gene therapies for neurodegenerative diseases.

Longitudinal Assessment of Lipoprotein(a) Levels in Perinatally HIV-Infected Children and Adolescents.

HIV is an independent risk factor of cardiovascular disease (CVD); therefore, perinatally HIV-infected (PHIV) children potentially have a greater CVD risk at older age. Lipoprotein(a) (Lp(a)) is an established risk factor for CVD in the general population. To evaluate a potential increased CVD risk for PHIV children, we determined their lipid profiles including Lp(a). In the first substudy, we assessed the lipid profiles of 36 PHIV children visiting the outpatient clinic in Amsterdam between 2012 and 2020. In the second substudy, we enrolled 21 PHIV adolescents and 23 controls matched for age, sex and ethnic background on two occasions with a mean follow-up time of 4.6 years. We assessed trends of lipid profiles and their determinants, including patient and disease characteristics, using mixed models. In the first substudy, the majority of PHIV children were Black (92%) with a median age of 8.0y (5.7-10.8) at first assessment. Persistent elevated Lp(a) levels were present in 21/36 (58%) children (median: 374 mg/L (209-747); cut off = 300). In the second substudy, the median age of PHIV adolescents was 17.5y (15.5-20.7) and of matched controls 16.4y (15.8-19.5) at the second assessment. We found comparable lipid profiles between groups. In both studies, increases in LDL-cholesterol and total cholesterol were associated with higher Lp(a) levels. A majority of PHIV children and adolescents exhibited elevated Lp(a) levels, probably associated with ethnic background. Nonetheless, these elevated Lp(a) levels may additionally contribute to an increased CVD risk.

PhP.B Enhanced Adeno-Associated Virus Mediated-Expression Following Systemic Delivery or Direct Brain Administration.

Of the adeno-associated viruses (AAVs), AAV9 is known for its capability to cross the blood-brain barrier (BBB) and can, therefore, be used as a noninvasive method to target the central nervous system. Furthermore, the addition of the peptide PhP.B to AAV9 increases its transduction across the BBB by 40-fold. Another neurotropic serotype, AAV5, has been shown as a gene therapeutic delivery vehicle to ameliorate several neurodegenerative diseases in preclinical models, but its administration requires invasive surgery. In this study, AAV9-PhP.B and AAV5-PhP.B were designed and produced in an insect cell-based system. To AAV9, the PhP.B peptide TLAVPFK was added, whereas in AAV5-PhP.B (AQTLAVPFKAQAQ), with AQ-AQAQ sequences used to swap with the corresponding sequence of AAV5. The addition of PhP.B to AAV5 did not affect its capacity to cross the mouse BBB, while increased transduction of liver tissue was observed. Then, intravenous (IV) and intrastriatal (IStr) delivery of AAV9-PhP.B and AAV5 were compared. For AAV9-PhP.B, similar transduction and expression levels were achieved in the striatum and cortex, irrespective of the delivery method used. IStr administration of AAV5 resulted in significantly higher amounts of vector DNA and therapeutic miRNA in the target regions such as striatum and cortex when compared with an IV administration of AAV9-PhP.B. These results illustrate the challenge in developing a vector that can be delivered noninvasively while achieving a transduction level similar to that of direct administration of AAV5. Thus, for therapeutic miRNA delivery with high local expression requirements, intraparenchymal delivery of AAV5 is preferred, whereas a humanized AAV9-PhP.B may be useful when widespread brain (and peripheral) transduction is needed.

Cerebral Organoids: A Human Model for AAV Capsid Selection and Therapeutic Transgene Efficacy in the Brain.

The development of gene therapies for central nervous system disorders is challenging because it is difficult to translate preclinical data from current in vitro and in vivo models to the clinic. Therefore, we developed induced pluripotent stem cell (iPSC)-derived cerebral organoids as a model for recombinant adeno-associated virus (rAAV) capsid selection and for testing efficacy of AAV-based gene therapy in a human context. Cerebral organoids are physiological 3D structures that better recapitulate the human brain compared with 2D cell lines. To validate the model, we compared the transduction efficiency and distribution of two commonly used AAV serotypes (rAAV5 and rAAV9). In cerebral organoids, transduction with rAAV5 led to higher levels of vector DNA, transgenic mRNA, and protein expression as compared with rAAV9. The superior transduction of rAAV5 was replicated in iPSC-derived neuronal cells. Furthermore, rAAV5-mediated delivery of a human sequence-specific engineered microRNA to cerebral organoids led to a lower expression of its target ataxin-3. Our studies provide a new tool for selecting and deselecting AAV serotypes, and for demonstrating therapeutic efficacy of transgenes in a human context. Implementing cerebral organoids during gene therapy development could reduce the usage of animal models and improve translation to the clinic.

Bone morphogenetic protein signaling suppresses tumorigenesis at gastric epithelial transition zones in mice.

Bone morphogenetic protein (BMP) signaling is known to suppress oncogenesis in the small and large intestine of mice and humans. We examined the role of Bmpr1a signaling in the stomach. On conditional inactivation of Bmpr1a, mice developed neoplastic lesions specifically in the squamocolumnar and gastrointestinal transition zones. We hypothesized that the regulation of epithelial cell fate may be less well defined in these junctional zones than in the adjacent epithelium and found that the mucosa at the squamocolumnar junction in mice shows a lack of differentiated fundic gland cell types and that foveolar cells at the gastrointestinal junctional zone lack expression of the foveolar cell marker Muc5ac. Precursor cell proliferation in both transition zones was higher than in the surrounding epithelium. Our data show that BMP signaling through Bmpr1a suppresses tumorigenesis at gastric epithelial junctional zones that are distinct from the adjacent gastric epithelium in both cellular differentiation and proliferation.

Immunoadsorption enables successful rAAV5-mediated repeated hepatic gene delivery in nonhuman primates.

Adeno-associated virus (AAV)-based liver gene therapy has been shown to be clinically successful. However, the presence of circulating neutralizing antibodies (NABs) against AAV vector capsids remains a major challenge as it may prevent successful transduction of the target cells. Therefore, there is a need to develop strategies that would enable AAV-mediated gene delivery to patients with preexisting anti-AAV NABs. In the current study, the feasibility of using an immunoadsorption (IA) procedure for repeated, liver-targeted gene delivery in nonhuman primates was explored. The animals were administered IV with recombinant AAV5 (rAAV5) carrying the reporter gene human secreted embryonic alkaline phosphatase (hSEAP). Seven weeks after the first rAAV treatment, all of the animals were readministered with rAAV5 carrying the therapeutic hemophilia B gene human factor IX (hFIX). Half of the animals administered with rAAV5-hSEAP underwent IA prior to the second rAAV5 exposure. The transduction efficacies of rAAV5-hSEAP and rAAV5-hFIX were assessed by measuring the levels of hSEAP and hFIX proteins. Although no hFIX was detected after rAAV5-hFIX readministration without prior IA, all animals submitted to IA showed therapeutic levels of hFIX expression, and a threshold of anti-AAV5 NAB levels compatible with successful readministration was demonstrated. In summary, our data demonstrate that the use of a clinically applicable IA procedure enables successful readministration of an rAAV5-based gene transfer in a clinically relevant animal model. Finally, the analysis of anti-AAV NAB levels in human subjects submitted to IA confirmed the safety and efficacy of the procedure to reduce anti-AAV NABs. Furthermore, clinical translation was assessed using an immunoglobulin G assay as surrogate.

Enhanced Factor IX Activity following Administration of AAV5-R338L "Padua" Factor IX versus AAV5 WT Human Factor IX in NHPs.

Gene therapy for severe hemophilia B is advancing and offers sustained disease amelioration with a single treatment. We have reported the efficacy and safety of AMT-060, an investigational gene therapy comprising an adeno-associated virus serotype 5 capsid encapsidating the codon-optimized wild-type human factor IX (WT hFIX) gene with a liver-specific promoter, in patients with severe hemophilia B. Treatment with 2 × 1013 gc/kg AMT-060 showed sustained and durable FIX activity of 3%-13% and a substantial reduction in spontaneous bleeds without T cell-mediated hepatoxicity. To achieve higher FIX activity, we modified AMT-060 to encode the R338L "Padua" FIX variant that has increased specific activity (AMT-061). We report the safety and increased FIX activity of AMT-061 in non-human primates. Animals (n = 3/group) received intravenous AMT-060 (5 × 1012 gc/kg), AMT-061 (ranging from 5 × 1011 to 9 × 1013 gc/kg), or vehicle. Human FIX protein expression, FIX activity, and coagulation markers including D-dimer and thrombin-antithrombin complexes were measured. At equal doses, AMT-060 and AMT-061 resulted in similar human FIX protein expression, but FIX activity was 6.5-fold enhanced using AMT-061. Both vectors show similar safety and transduction profiles. Thus, AMT-061 holds great promise as a more potent FIX replacement gene therapy with a favorable safety profile.

AAV5-miHTT Lowers Huntingtin mRNA and Protein without Off-Target Effects in Patient-Derived Neuronal Cultures and Astrocytes.

Huntington disease (HD) is a fatal neurodegenerative genetic disorder, thought to reflect a toxic gain of function in huntingtin (Htt) protein. Adeno-associated viral vector serotype 5 (AAV5)- microRNA targeting huntingtin (miHTT) is a HD gene-therapy candidate that efficiently lowers HTT using RNAi. This study analyzed the efficacy and potential for off-target effects with AAV5-miHTT in neuronal and astrocyte cell cultures differentiated from induced pluripotent stem cells (iPSCs) from two individuals with HD (HD71 and HD180). One-time AAV5-miHTT treatment significantly reduced human HTT mRNA by 57% and Htt protein by 68% in neurons. Small RNA sequencing showed that mature miHTT was processed correctly without off-target passenger strand. No cellular microRNAs were dysregulated, indicating that endogenous RNAi machinery was unaffected by miHTT overexpression. qPCR validation of in silico-predicted off-target transcripts, next-generation sequencing, and pathway analysis confirmed absence of dysregulated genes due to sequence homology or seed-sequence activity of miHTT. Minor effects on gene expression were observed in both AAV5-miHTT and AAV5-GFP-treated samples, suggesting that they were due to viral transduction rather than miHTT. This study confirms the efficacy of AAV5-miHTT in HD patient iPSC-derived neuronal cultures and lack of off-target effects in gene expression and regulation in neuronal cells and astrocytes.

Therapeutic hFIX Activity Achieved after Single AAV5-hFIX Treatment in Hemophilia B Patients and NHPs with Pre-existing Anti-AAV5 NABs.

Currently, individuals with pre-existing neutralizing antibodies (NABs) against adeno-associated virus (AAV) above titer of 5 are excluded from systemic AAV-based clinical trials. In this study we explored the impact of pre-existing anti-AAV5 NABs on the efficacy of AAV5-based gene therapy. AMT-060 (AAV5-human FIX) was evaluated in 10 adults with hemophilia B who tested negative for pre-existing anti-AAV5 NABs using a GFP-based assay. In this study, using a more sensitive luciferase-based assay, we show that 3 of those 10 patients tested positive for anti-AAV5 NABs. However, no relationship was observed between the presence of pre-treatment anti-AAV5 NABs and the therapeutic efficacy of AMT-060. Further studies in non-human primates (NHPs) showed that AAV5 transduction efficacy was similar following AMT-060 treatment, irrespective of the pre-existing anti-AAV5 NABs titers. We show that therapeutic efficacy of AAV5-mediated gene therapy was achieved in humans with pre-existing anti-AAV5 NABs titers up to 340. Whereas in NHPs circulating human factor IX (hFIX) protein was achieved, at a level therapeutic in humans, with pre-existing anti-AAV5 NABs up to 1030. Based on those results, no patients were excluded from the AMT-061 (AAV5-hFIX-Padua) phase IIb clinical trial (n = 3). All three subjects presented pre-existing anti-AAV5 NABs, yet had therapeutic hFIX activity after AMT-061 administration.

AAV5-miHTT Gene Therapy Demonstrates Sustained Huntingtin Lowering and Functional Improvement in Huntington Disease Mouse Models.

Huntington disease (HD) is a fatal neurodegenerative disorder caused by an autosomal dominant CAG repeat expansion in the huntingtin (HTT) gene. The translated expanded polyglutamine repeat in the HTT protein is known to cause toxic gain of function. We showed previously that strong HTT lowering prevented neuronal dysfunction in HD rodents and minipigs after single intracranial injection of adeno-associated viral vector serotype 5 expressing a microRNA targeting human HTT (AAV5-miHTT). To evaluate long-term efficacy, AAV5-miHTT was injected into the striatum of knockin Q175 HD mice, and the mice were sacrificed 12 months post-injection. AAV5-miHTT caused a dose-dependent and sustained HTT protein reduction with subsequent suppression of mutant HTT aggregate formation in the striatum and cortex. Functional proof of concept was shown in transgenic R6/2 HD mice. Eight weeks after AAV5-miHTT treatment, a significant improvement in motor coordination on the rotarod was observed. Survival analysis showed that a single AAV5-miHTT treatment resulted in a significant 4-week increase in median survival compared with vehicle-treated R6/2 HD mice. The combination of long-term HTT lowering, reduction in aggregation, prevention of neuronal dysfunction, alleviation of HD-like symptoms, and beneficial survival observed in HD rodents treated with AAV5-miHTT supports the continued development of HTT-lowering gene therapies for HD.

Artificial MicroRNAs Targeting C9orf72 Can Reduce Accumulation of Intra-nuclear Transcripts in ALS and FTD Patients.

The most common pathogenic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic GGGGCC (G4C2) repeat in the chromosome 9 open reading frame 72 (C9orf72) gene. Cellular toxicity due to RNA foci and dipeptide repeat (DPR) proteins produced by the sense and antisense repeat-containing transcripts is thought to underlie the pathogenesis of both diseases. RNA sequencing (RNA-seq) data of C9orf72-ALS patients and controls were analyzed to better understand the sequence conservation of C9orf72 in patients. MicroRNAs were developed in conserved regions to silence C9orf72 (miC), and the feasibility of different silencing approaches was demonstrated in reporter overexpression systems. In addition, we demonstrated the feasibility of a bidirectional targeting approach by expressing two concatenated miC hairpins. The efficacy of miC was confirmed by the reduction of endogenously expressed C9orf72 mRNA, in both nucleus and cytoplasm, and an ∼50% reduction of nuclear RNA foci in (G4C2)44-expressing cells. Ultimately, two miC candidates were incorporated in adeno-associated virus vector serotype 5 (AAV5), and silencing of C9orf72 was demonstrated in HEK293T cells and induced pluripotent stem cell (iPSC)-derived neurons. These data support the feasibility of microRNA (miRNA)-based and AAV-delivered gene therapy that could alleviate the gain of toxicity seen in ALS and FTD patients.

Targeting RNA-Mediated Toxicity in C9orf72 ALS and/or FTD by RNAi-Based Gene Therapy.

A hexanucleotide GGGGCC expansion in intron 1 of chromosome 9 open reading frame 72 (C9orf72) gene is the most frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The corresponding repeat-containing sense and antisense transcripts cause a gain of toxicity through the accumulation of RNA foci in the nucleus and deposition of dipeptide-repeat (DPR) proteins in the cytoplasm of the affected cells. We have previously reported on the potential of engineered artificial anti-C9orf72-targeting miRNAs (miC) targeting C9orf72 to reduce the gain of toxicity caused by the repeat-containing transcripts. In the current study, we tested the silencing efficacy of adeno-associated virus (AAV)5-miC in human-derived induced pluripotent stem cell (iPSC) neurons and in an ALS mouse model. We demonstrated that AAV5-miC transduces different types of neuronal cells and can reduce the accumulation of repeat-containing C9orf72 transcripts. Additionally, we demonstrated silencing of C9orf72 in both the nucleus and cytoplasm, which has an added value for the treatment of ALS and/or FTD patients. A proof of concept in an ALS mouse model demonstrated the significant reduction in repeat-containing C9orf72 transcripts and RNA foci after treatment. Taken together, these findings support the feasibility of a gene therapy for ALS and FTD based on the reduction in toxicity caused by the repeat-containing C9orf72 transcripts.