Guangping Gao

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.

Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras

Recombinant adeno-associated virus (rAAV)-based gene therapy has entered a phase of clinical translation and commercialization. Despite this progress, vector integrity following production is often overlooked. Compromised vectors may negatively impact therapeutic efficacy and safety. Using single molecule, real-time (SMRT) sequencing, we can comprehensively profile packaged genomes as a single intact molecule and directly assess vector integrity without extensive preparation. We have exploited this methodology to profile all heterogeneic populations of self-complementary AAV genomes via bioinformatics pipelines and have coined this approach AAV-genome population sequencing (AAV-GPseq). The approach can reveal the relative distribution of truncated genomes versus full-length genomes in vector preparations. Preparations that seemingly show high genome homogeneity by gel electrophoresis are revealed to consist of less than 50% full-length species. With AAV-GPseq, we can also detect many reverse-packaged genomes that encompass sequences originating from plasmid backbone, as well as sequences from packaging and helper plasmids. Finally, we detect host-cell genomic sequences that are chimeric with inverted terminal repeat (ITR)-containing vector sequences. We show that vector populations can contain between 1.3% and 2.3% of this type of undesirable genome. These discoveries redefine quality control standards for viral vector preparations and highlight the degree of foreign products in rAAV-based therapeutic vectors.

Adeno-Associated Virus Neutralizing Antibodies in Large Animals and Their Impact on Brain Intraparenchymal Gene Transfer

Pre-existing neutralizing antibody (NAb) against adeno-associated virus (AAV) commonly found in primates is a major host barrier that can severely compromise in vivo gene transfer by AAV vectors. To achieve proof-of-concept success in clinical development of recombinant AAV (rAAV)-based in vivo gene therapy, it is crucial to consider the potential interference of NAb and to enroll serologically compatible study subjects. In this study, we report a large AAV NAb dataset comprising multiple large animal species and AAV serotypes and compare two NAb assays based on in vitro or in vivo transduction inhibition, respectively. Together with previously published AAV seroepidemiology studies, these data can serve as a reference for selecting suitable serotypes, study subjects of large animal species, and potentially human patients for rAAV treatment. In addition, we modeled the intrathalamus rAAV9 delivery in the presence of circulating anti-AAV9 NAb generated by either pre-immunization or passive transfer of NAb-positive large animal serum to mice. The data showed that circulating NAb may not be the sole determinant to inhibit brain transduction. Other aspects of pre-existing AAV immunity following natural infection or rAAV administration may be further studied to establish a more accurate inclusion criterion for clinical studies employing intraparenchymal rAAV9 injections.

MicroRNA-96 Promotes Schistosomiasis Hepatic Fibrosis in mice by suppressing Smad7

Infection with Schistosoma causes aberrant expression of host microRNAs (miRNAs), and normalizing the levels of dysregulated miRNAs can attenuate pathology. Here, we show that the host miRNA, miR-96, is markedly upregulated during the progression of hepatic schistosomiasis. We demonstrate that elevation of miR-96 induces hepatic fibrosis in infected mice by suppressing the expression of its target gene, Smad7. We show that infection with Schistosoma induces the expression of transforming growth factor β1 (TGF-β1), which in turn upregulates the expression of miR-96 through SMAD2/3-DROSHA-mediated post-transcriptional regulation. Furthermore, inhibition of miR-96 with recombinant adeno-associated virus 8 (rAAV8)-mediated delivery of Tough Decoy RNAs in mice attenuated hepatic fibrosis and prevented lethality following schistosome infection. Taken together, our data highlight the potential for rAAV8-mediated inhibition of miR-96 as a therapeutic strategy to treat hepatic schistosomiasis.

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.

9. rAAV Designs Harboring DNA Secondary Structures with High Thermal Stabilities Produce Heterogenic Viral Genome Populations

It has been known for more than a decade that AAV genomes in ssAAV particles are heterogeneous, and can contain smaller than unit-length molecules to varying degrees. However, the cause underlying this phenomenon remained unknown. Our earlier work demonstrated that short DNA hairpin (shDNA) sequences designed into rAAV vectors, resulted in truncated genomes through a template-switching mechanism during AAV genome replication. Importantly, the knowledge we have gained about shDNA-mediated genomic truncation has helped to improve shRNA-cassette design. Specifically, we have found a correlation between the thermal stability of shDNA structures with the prevalence of truncation events. By introducing point mutations into the passenger strand of shRNA sequences to create DNA bulges within shDNAs in scAAV vectors, we found that lowering the thermal stability of DNA hairpins lowered the proportion of truncated to complete genomes. In addition, we found that scAAV vectors incorporated with pri-miRNA transgenes, which contain natural bulges in their stem-loop structures, also produced fewer truncated genomes, relative to shDNA-rAAV constructs. By embedding the guide strand of small RNAs into pri-miRNA scaffolds, we have defined critical improvements to the genomic integrity of rAAV vectors expressing small RNAs. Furthermore, we developed a method, named AAV-GPSeq (AAV genome populations sequencing), to directly sequence whole vector genome populations from purified rAAVs using the PacBio platform for high-throughput sequencing. We discovered through this methodology that truncation events can originate from palindromic sequences and inverted repeats that reside in the expression cassette elements of widely used ssAAV and scAAV vector designs (i.e. transcriptional regulatory elements, transgene sequences, and post-transcriptional regulatory regions). The resulting diversity of genomic truncations produces populations of packaged virions with variable transgene efficacies, and gives us the first insights into the phenomenon of rAAV heterogeneity. By examining the genomes from rAAV vectors harboring shDNAs, pri-miRNA fragments, and palindromic/inverted repeat sequences, we now highlight the importance of DNA secondary structure on vector design and genomic heterogeneity in packaged viral vectors. Improvements upon quality control standards are thus necessary and critical to efficacious and safe clinical uses for rAAV as a biomedicine. Further implications for our novel findings towards understanding AAV replication, and new considerations for future therapeutic rAAV vector designs will be discussed. To improve the homogeneity of clinical rAAV vectors, we are optimizing the vector production procedure, testing non-palindrome promoters, changing the codon usage in transgenes to lower the thermostability of rAAV genomes, and modifying rAAV packaging cell lines to minimize template-switching events to produce more intact rAAV genomes.