Dominic J. Gessler

Global CNS transduction of adult mice by intravenously delivered rAAVrh.8 and rAAVrh.10 and nonhuman primates by rAAVrh.10

Some recombinant adeno-associated viruses (rAAVs) can cross the neonatal blood-brain barrier (BBB) and efficiently transduce cells of the central nervous system (CNS). However, in the adult CNS, transduction levels by systemically delivered rAAVs are significantly reduced, limiting their potential for CNS gene therapy. Here, we characterized 12 different rAAVEGFPs in the adult mouse CNS following intravenous delivery. We show that the capability of crossing the adult BBB and achieving widespread CNS transduction is a common character of AAV serotypes tested. Of note, rAAVrh.8 is the leading vector for robust global transduction of glial and neuronal cell types in regions of clinical importance such as cortex, caudate-putamen, hippocampus, corpus callosum, and substantia nigra. It also displays reduced peripheral tissue tropism compared to other leading vectors. Additionally, we evaluated rAAVrh.10 with and without microRNA (miRNA)-regulated expressional detargeting from peripheral tissues for systemic gene delivery to the CNS in marmosets. Our results indicate that rAAVrh.8, along with rh.10 and 9, hold the best promise for developing novel therapeutic strategies to treat neurological diseases in the adult patient population. Additionally, systemically delivered rAAVrh.10 can transduce the CNS efficiently, and its transgene expression can be limited in the periphery by endogenous miRNAs in adult marmosets.

Redirecting N-acetylaspartate metabolism in the central nervous system normalizes myelination and rescues Canavan disease

Canavan disease (CD) is a debilitating and lethal leukodystrophy caused by mutations in the aspartoacylase (ASPA) gene and the resulting defect in N-acetylaspartate (NAA) metabolism in the CNS and peripheral tissues. Recombinant adeno-associated virus (rAAV) has the ability to cross the blood-brain barrier and widely transduce the CNS. We developed a rAAV-based and optimized gene replacement therapy, which achieves early, complete, and sustained rescue of the lethal disease phenotype in CD mice. Our treatment results in a super-mouse phenotype, increasing motor performance of treated CD mice beyond that of WT control mice. We demonstrate that this rescue is oligodendrocyte independent, and that gene correction in astrocytes is sufficient, suggesting that the establishment of an astrocyte-based alternative metabolic sink for NAA is a key mechanism for efficacious disease rescue and the super-mouse phenotype. Importantly, the use of clinically translatable high-field imaging tools enables the noninvasive monitoring and prediction of therapeutic outcomes for CD and might enable further investigation of NAA-related cognitive function.

Gene Therapy for the Treatment of Neurological Disorders: Metabolic Disorders.

Metabolic disorders comprise a large group of heterogeneous diseases ranging from very prevalent diseases such as diabetes mellitus to rare genetic disorders like Canavan Disease. Whether either of these diseases is amendable by gene therapy depends to a large degree on the knowledge of their pathomechanism, availability of the therapeutic gene, vector selection, and availability of suitable animal models. In this book chapter, we review three metabolic disorders of the central nervous system (CNS; Canavan Disease, Niemann-Pick disease and Phenylketonuria) to give examples for primary and secondary metabolic disorders of the brain and the attempts that have been made to use adeno-associated virus (AAV) based gene therapy for treatment. Finally, we highlight commonalities and obstacles in the development of gene therapy for metabolic disorders of the CNS exemplified by those three diseases.

Adeno-associated virus serotype rh.10 displays strong muscle tropism following intraperitoneal delivery

Recombinant adeno-associated virus (rAAV) is an attractive tool for basic science and translational medicine including gene therapy, due to the versatility in its cell and organ transduction. Previous work indicates that rAAV transduction patterns are highly dependent on route of administration. Based on this relationship, we hypothesized that intraperitoneal (IP) administration of rAAV produces unique patterns of tissue tropism. To test this hypothesis, we investigated the transduction efficiency of 12 rAAV serotypes carrying an enhanced green fluorescent protein (EGFP) reporter gene in a panel of 12 organs after IP injection. Our data suggest that IP administration emphasizes transduction patterns that are different from previously reported intravascular delivery methods. Using this approach, rAAV efficiently transduces the liver, pancreas, skeletal muscle, heart and diaphragm without causing significant histopathological changes. Of note, rAAVrh.10 showed excellent muscle transduction following IP administration, highlighting its potential as a new muscle-targeting vector.

Cas9-mediated allelic exchange repairs compound heterozygous recessive mutations in mice

We report a genome-editing strategy to correct compound heterozygous mutations, a common genotype in patients with recessive genetic disorders. Adeno-associated viral vector delivery of Cas9 and guide RNA induces allelic exchange and rescues the disease phenotype in mouse models of hereditary tyrosinemia type I and mucopolysaccharidosis type I. This approach recombines non-mutated genetic information present in two heterozygous alleles into one functional allele without using donor DNA templates.We report a genome-editing strategy to correct compound heterozygous mutations, a common genotype in patients with recessive genetic disorders. Adeno-associated viral vector delivery of Cas9 and guide RNA induces allelic exchange and rescues the disease phenotype in mouse models of hereditary tyrosinemia type I and mucopolysaccharidosis type I. This approach recombines non-mutated genetic information present in two heterozygous alleles into one functional allele without using donor DNA templates.

A Rationally Engineered Capsid Variant of AAV9 for Systemic CNS-Directed and Peripheral Tissue-Detargeted Gene Delivery in Neonates

Adeno-associated virus (AAV) has provided the gene therapy field with the most powerful in vivo gene delivery vector to realize safe, efficacious, and sustainable therapeutic gene expression. Because many clinically relevant properties of AAV-based vectors are governed by the capsid, much research effort has been devoted to the development of AAV capsids for desired features. Here, we combine AAV capsid discovery from nature and rational engineering to report an AAV9 capsid variant, designated as AAV9.HR, which retains AAV9’s capability to traverse the blood-brain barrier and transduce neurons. This variant shows reduced transduction in peripheral tissues when delivered through intravascular (IV) injection into neonatal mice. Therefore, when IV AAV delivery is used to treat CNS diseases, AAV9.HR has the advantage of mitigating potential off-target effects in peripheral tissues compared to AAV9. We also demonstrate that AAV9.HR is suitable for peripheral tissue-detargeted CNS-directed gene therapy in a mouse model of a fatal pediatric leukodystrophy. In light of recent success with profiling diversified natural AAV capsid repertoires and the understanding of AAV capsid sequence-structure-function relationship, such a combinatory approach to AAV capsid development is expected to further improve vector targeting and expand the vector toolbox for therapeutic gene delivery.

Transcriptome Profiling of Neovascularized Corneas Reveals miR-204 as a Multi-target Biotherapy Deliverable by rAAVs

Corneal neovascularization (NV) is the major sight-threatening pathology caused by angiogenic stimuli. Current drugs that directly target pro-angiogenic factors to inhibit or reverse the disease require multiple rounds of administration and have limited efficacies. Here, we identify potential anti-angiogenic corneal microRNAs (miRNAs) and demonstrate a framework that employs discovered miRNAs as biotherapies deliverable by recombinant adeno-associated viruses (rAAVs). By querying differentially expressed miRNAs in neovascularized mouse corneas induced by alkali burn, we have revealed 39 miRNAs that are predicted to target more than 5,500 differentially expressed corneal mRNAs. Among these, we selected miR-204 and assessed its efficacy and therapeutic benefit for treating injured corneas. Our results show that delivery of miR-204 by rAAV normalizes multiple novel target genes and biological pathways to attenuate vascularization of injured mouse cornea. Importantly, this gene therapy treatment alternative is efficacious and safe for mitigating corneal NV. Overall, our work demonstrates the discovery of potential therapeutic miRNAs in corneal disorders and their translation into viable treatment alternatives.

733. Somatically Repairing Compound Heterozygous Recessive Mutations by Chromosomal Cut-and-Paste for In Vivo Gene Therapy

Patients affected by monogenic recessive genetic disorders often carry two different mutated alleles of the same gene, which is known as compound heterozygous. Theoretically, exchanging the genetic material between the two mutated alleles will reconstitute a mutation-free allele that can be therapeutic (FigureFigure). We hypothesized that generating DNA double-stranded breaks at the same location on both mutant alleles can induce allele exchange, reconstitute a mutation-free allele, and therefore yield therapeutic benefit. We first tested this hypothesis in a targeted knock-in mouse model that carries GFPN-term-intron-tdTomatoC-term and tdTomatoN-term-intron-GFPC-term expression cassettes, respectively, at the same genomic location on each copy of Chr 11. Therefore, allele exchange at the intronic region will reconstitute the full-length GFP and tdTomato, serving as a reporter system. We injected recombinant AAV (rAAV) vectors expressing SpCas9 and sgRNA targeting the intron into adult mice by tail vein injection. Five weeks later, we observed GFP and tdTomato fluorescence in cryosections of peripheral tissues including liver and heart, whereas there was no such fluorescence observed in the tissue samples from untreated mice, demonstrating that allele exchange occurred, and that the reconstituted alleles yielded protein expression. Furthermore, we generated mice that carry two different mutations of the Aspa gene as a compound heterozygous mouse model of Canavan disease. We treated these mice with rAAV vectors expressing SpCas9 and sgRNA targeting an intron between the two mutation sites. Three weeks after treatment, we detected reconstituted, mutation-free Aspa DNA sequence by allele-specific PCR and single-molecule, high-throughput DNA sequencing in the liver. The reconstituted Aspa allele carried insertion at the predicted SpCas9 cleavage site, indicating that the DNA allele exchange was mediated by the non-homologous end joining DNA repair pathway. We also observed allele exchange in mouse liver using the SaCas9 system. Evaluation of the therapeutic benefit following Cas9/sgRNA-mediated allele exchange in various compound heterozygous mouse models and patient cell lines is underway. The gene repairing strategy described here is a novel approach to tackling a broad range of autosomal recessive genetic disorders.View Large Image | Download PowerPoint Slide

157. High-Field In Vivo Neuroimaging to Determine CNS Gene Therapy Outcome and Probe Disease Pathomechanism

Gene therapy targeting the central nervous system (CNS) is one of the most challenging gene therapies due to the blood-brain barrier (BBB). However, recombinant adeno-associated virus (rAAV) has proved to be an excellent tool to target the CNS. Another obstacle in the setting of CNS gene therapy is the non-invasive evaluation of therapeutic outcome. While biopsies and sections of the CNS are the gold-standard to assess brain pathology and response to CNS gene therapy, the invasiveness and potentially associated complications limit its frequent use in pre-clinical as well as clinical studies. Thus, it is of no surprise that non-invasive monitoring of CNS gene therapy in vivo holds great promise for longitudinal and functional assessment of treatment response. We used high-field in vivo neuroimaging to monitor intravenously (i.v.) and intracerebroventricularly (i.c.v.) administered rAAV based CNS directed gene therapy in a mouse model of Canavan disease (CD). Characteristically, Canavan disease presents with a very high NAA peak detected by magnet resonance spectrometry (MRS) and hyper intensity on T2-weighted anatomic images using magnet resonance imaging (MRI). Consequently, we first determined the efficacy of our i.v. and i.c.v. gene therapy by those two means. In congruence with motor function and pathology data, both MRI and MRS alterations have been entirely normalized by gene therapy. Another characteristic neuropathological change on Canavan brain sections is the loss of white matter tracts, which is thought to explain neurological symptoms seen in Canavan disease patients. Thus, we hypothesized that diffusion tensor imaging (DTI) enables the assessment of white matter tract degeneration and recovery upon gene therapy without brain biopsies. Selecting thalamus and corpus callosum as regions of interest (ROI), DTI indeed shows a recovery of brain white matter integrity when utilizing 3rd generation Canavan gene therapy. Furthermore, our 3rd generation gene therapy converts this CD mouse model with the severest phenotype into “supermouse”, outperforming wild-type mouse on motor function testing. We hypothesized that functional connectivity identifies brain regions that not only show response to treatment but also indicates possible explanations for this enhanced phenotype. Using resting-state functional MRI (rs-fMRI), we show that treated CD mice have a functional connectivity pattern that is more similar to or even enhanced beyond what is seen in WT brain. This suggests facilitated inter-brain-region functional connectivity, which might provide a neural mechanism that sub-serves the observed enhanced motor function. Currently, we are investigating how the identified brain regions can promote increased motor function, and how high-field brain imaging can provide biomarkers to track the disorder and treatment response in a manner that would help facilitate the prediction of CNS directed gene therapy outcome. In summary, our data show that high-field in vivo neuroimaging is a valuable tool to monitor pre-clinical CNS gene therapy and pathology in detail, that it can provide insights into pathophysiology and that it has potential implications for the use in clinical trial outcome prediction and assessment.

739. rAAV Delivered MicroRNA Therapeutics Towards Efficacious Treatment of Corneal Neovascularization

Corneal diseases are the second major cause of blindness worldwide. Corneal neovascularization (NV) is one of the most common pathologies in corneal diseases, leading to vision loss or even blindness. However, even though different treatments for corneal NV are available, a safe and effective therapy remains to be an unmet medical challenge. MicroRNAs (miRNAs) play roles in regulating more than half of all protein-coding genes in mammals including those involved with angiogenesis. We hypothesize that modulating expression of angiogenesis related miRNAs might be an effective approach for treating corneal NV. To this end, we first set out to identify target miRNAs that play roles in corneal NV using Nanostring technologies and the classic alkali-burn induced corneal NV mouse model. Among 618 mouse miRNAs profiled, we found 35 up-regulated and 3 down-regulated miRNAs in the neovascularized corneas, which were confirmed by qRT-PCR. We selected miR-184 and miR-204, two miRNAs that were down-regulated > 10-folds in neovascularized corneas, as our candidate targets to test the concept of in vivo gene delivery for therapeutic miRNA to treat corneal NV. As recombinant adeno-associated virus (rAAV) holds promise for highly efficient and safe in vivo gene delivery to different target tissues in a serotype dependent manner, we opted to evaluate 14 serotypes of rAAV.EGFP for gene transfer efficiency in mouse corneas by eye drops (ED), intra-stromal (IS) and subconjunctival (SC) injections to search for the suitable vector(s) for delivering miRNA therapeutics to the corneas. Among the 4 leading serotype vectors (i.e. rAAVrh.8, rh.10, rh.39 and rh.43) identified, we chose rAAVrh.10 for overexpressing pri miR-184 and pri miR-204 in the corneas with alkali-burn induced corneal NV. rAAVrh.10-mediated overexpression of miR-184 or miR-204 by IS and SC injections but not ED efficiently inhibited angiogenesis and was efficacious and safe for either prevention (IS) or treatment (SC) of corneal NV. We further invested potential target genes and pathways to elucidate the anti-angiogenic mechanism of these two miRNA therapeutics. We revealed that the anti-angiogenic effect of rAAV.pri-miR-184 was achieved by targeting Fzd4 gene, thus suppressing the canonical Wnt signaling, a well-known pathway playing vital roles in angiogenesis; while rAAV.pri-miR-204 exerted its anti-angiogenic effect by targeting angiopoietin-1 gene (Angpt1), a well-acknowledged pro-angiogenic factor. Meanwhile, the transcriptome analysis is under the way to search for novel gene targets of the miRNA therapeutics. Our study has great clinical relevance and demonstrated, for the first time, that miRNA-targeted novel therapeutics that can be delivered as either rAAV or synthetic nucleic acid drugs (i.e. miRNA mimics and antagomir) might offer an additional efficacious and safe clinical option for treating corneal NV and other angiogenesis-related diseases including age-related macular edema (AMD) and cancer.