Shaoyong Li

Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses.

CRISPR/Cas9 derived from the bacterial adaptive immunity pathway is a powerful tool for genome editing, but the safety profiles of in vivo delivered Cas9 (including host immune responses to the bacterial Cas9 protein) have not been comprehensively investigated in model organisms. Nonalcoholic steatohepatitis (NASH) is a prevalent human liver disease characterized by excessive fat accumulation in the liver. In this study, we used adenovirus (Ad) vector to deliver a Streptococcus pyogenes-derived Cas9 system (SpCas9) targeting Pten, a gene involved in NASH and a negative regulator of the PI3K-AKT pathway, in mouse liver. We found that the Ad vector mediated efficient Pten gene editing even in the presence of typical Ad vector-associated immunotoxicity in the liver. Four months after vector infusion, mice receiving the Pten gene-editing Ad vector showed massive hepatomegaly and features of NASH, consistent with the phenotypes following Cre-loxP-induced Pten deficiency in mouse liver. We also detected induction of humoral immunity against SpCas9 and the potential presence of an SpCas9-specific cellular immune response. Our findings provide a strategy to model human liver diseases in mice and highlight the importance considering Cas9-specific immune responses in future translational studies involving in vivo delivery of CRISPR/Cas9.

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.

Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects

Empty virions are inadvertent by-products of recombinant adeno-associated virus (rAAV) packaging process, resulting in vector lots with mixtures of full and empty virions at variable ratios. Impact of empty virions on the efficiency and side effects of rAAV transduction has not been well characterized. Here, we generated partially and completely empty AAV8 virions, fully packaged rAAV8 lots, and mixtures of empty and fully packaged virions with variable ratios of empty virions. The aforementioned dosing formulations of rAAV8 expressing either cellular (EGFP (enhanced green fluorescent protein) or nuclear-targeted (n) LacZ) or secreted (human α1-antitrypsin (hA1AT)) reporter genes were intravenously injected into two different mouse strains, followed by analyses of transgene expressions and serum alanine aminotransferase (ALT) levels at different time points. We found that addition of empty particles to the fixed doses of rAAV8 preparations repressed liver transduction up to 64% (serum hA1AT) and 44% (nLacZ) in C57BL/6 mice, respectively. The similar trend in inhibiting EGFP expression together with concurrent elevations of serum ALT levels were observed in the BALB/c mice, indicating that empty particles may also exacerbate side effects of rAAV8 EGFP transduction. Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.

Efficient and Targeted Transduction of Nonhuman Primate Liver With Systemically Delivered Optimized AAV3B Vectors

Recombinant adeno-associated virus serotype 3B (rAAV3B) can transduce cultured human liver cancer cells and primary human hepatocytes efficiently. Serine (S)- and threonine (T)-directed capsid modifications further augment its transduction efficiency. Systemically delivered capsid-optimized rAAV3B vectors can specifically target cancer cells in a human liver cancer xenograft model, suggesting their potential use for human liver-directed gene therapy. Here, we compared transduction efficiencies of AAV3B and AAV8 vectors in cultured primary human hepatocytes and cancer cells as well as in human and mouse hepatocytes in a human liver xenograft NSG-PiZ mouse model. We also examined the safety and transduction efficacy of wild-type (WT) and capsid-optimized rAAV3B in the livers of nonhuman primates (NHPs). Intravenously delivered S663V+T492V (ST)-modified self-complementary (sc) AAV3B-EGFP vectors led to liver-targeted robust enhanced green fluorescence protein (EGFP) expression in NHPs without apparent hepatotoxicity. Intravenous injections of both WT and ST-modified rAAV3B.ST-rhCG vectors also generated stable super-physiological levels of rhesus chorionic gonadotropin (rhCG) in NHPs. The vector genome predominantly targeted the liver. Clinical chemistry and histopathology examinations showed no apparent vector-related toxicity. Our studies should be important and informative for clinical development of optimized AAV3B vectors for human liver-directed gene therapy.

Recombinant Adeno-Associated Virus Integration Sites in Murine Liver After Ornithine Transcarbamylase Gene Correction

Recombinant adeno-associated viruses (rAAVs) have been tested in humans and other large mammals without adverse events. However, one study of mucopolysaccharidosis VII correction in mice showed repeated integration of rAAV in cells from hepatocellular carcinoma (HCC) in the Dlk1-Dio3 locus, suggesting possible insertional mutagenesis. In contrast, another study found no association of rAAV integration with HCC, raising questions about the generality of associations between liver transformation and integration at Dlk1-Dio3. Here we report that in rAAV-treated ornithine transcarbamylase (Otc)-deficient mice, four examples of integration sites in Dlk1-Dio3 could be detected in specimens from liver nodule/tumors, confirming previous studies of rAAV integration in the Dlk1-Dio3 locus in the setting of another murine model of metabolic disease. In one case, the integrated vector was verified to be present at about one copy per cell, consistent with clonal expansion. Another verified integration site in liver nodule/tumor tissue near the Tax1bp1 gene was also detected at about one copy per cell. The Dlk1-Dio3 region has also been implicated in human HCC and so warrants careful monitoring in ongoing human clinical trials with rAAV vectors.

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.

81. Downregulation of Immune Responsive Chemokines Are Associated AAV8-Mediated Transgene Immune Tolerance

Recombinant adeno-associated virus (rAAV)-mediated liver transduction can induce transgene product-specific immune tolerance, which can be further extended to the systemic tolerance. Because of such tolerogenic properties and its physiological functions, the liver has been a preferred target organ for AAV- gene therapy. The hepatic regulatory T cells (Tregs) are regarded as the major factor that contributes the immune tolerance in the liver. Here, we first tested if AAV capsid plays a role in inducing systemic immune tolerance. To this end, we used highly immunogenic Ovalbumin (OVA) as a model antigen expressed under the direction of a strong ubiquitous chicken β-actin (CB) promoter and packaged the vector with both AAV1 and AAV8 capsids for in vivo delivery to C57BL/6 mice. Compared to AAV1, AAV8 achieved significantly higher OVA expression without IgG production, regardless of intravenous (IV) or intramuscular (IM) injection. In addition, IV and IM delivered AAV1. OVA both generated OVA-specific CD8+ T cells in spleen, while AAV8-mediated deliveries blunted CD8+ T cell response and, instead, provoked B- and T-Reg responses. In an attempt to explore other possible cellular mechanism(s) led to hepatic tolerogenic properties, we profiled expression of innate and adaptive immune responsive genes that are possibly associated with liver immune tolerance in the context of rAAV transduction. We revealed that rAAV1.CB. OVA injections significantly elevated IL1a and CXCL1 gene expression as compared to the PBS and rAAV8 groups. As rAAV8 is more liver tropic than rAAVl, we hypothesized that hepatic OVA expression is essential to inducing immune tolerance. We tested this hypothesis by creating rAAV8.CB. OVA vectors (+/-) the binding sites for the liver enriched miR-122 and IV dosing C57BL/6 mice with the vectors at 4 doses (1×109, 1010, 1011 and 1012 gcs/mouse). We confirmed that miR-122 indeed effectively inactivated OVA expression from rAAV8.CB. OVA(+)MiR-122BS transduced liver; however, rAAV8.CB. OVA(-) miR122BS but not rAAV1CBOVA achieved sustained OVA expression in a dose dependent manner without eliciting OVA specific IgG. Finally, considering that the route of rAAV administration may affect transgene expression and immunity, we IM injected C57BL/6 mice with AAV8.CB. OVA (+/-) miR122BS vectors and found that AAV8.CB. OVA (-)miR-122BS expressed OVA efficiently without OVA-specific IgG production. In contrast, just as rAAV1.CB. OVA did, rAAV8.CB. OVA(+) miR-122BS produced significant OVA-specific IgG without OVA expression. Interestingly, IM delivered rAAV8.CB. OVA(+) miR-122 group expressed significantly higher levels of CXCL1, Myeloperoxidase(Mpo) and CCL5 in liver, as compared the PBS and rAAV8OVA(-)miR-122 groups as detected by qRT-PCR. Our data suggest that the downregulation of CXCL1, Mpo, CCL5 and IL1α in the liver are involved in the immune tolerance to rAAV8 delivered transgene. Those cytokines may represent therapeutic targets against autoimmune diseases.

457. Both Regulatory T and B Cells May Play Roles in Systemic Immune Tolerance Induced by rAAV-Mediated Gene Transfer to Liver

The intramuscular delivery of vectored antibody by recombinant adeno-associated virus (rAAV) holds promise for preventing and treating emerging infectious diseases. Recent studies in nonhuman primates suggested that a single intramuscular injection of rAAV1 expressing anti-SIV neutralizing antibody provided long-term protection from SIV infection. However, some of NHP recipients generated anti-antibody responses that eliminate neutralizing activity of vectored antibodies. We exploit different strategies to engineer AAV vectors that can surmount such anti-antibody responses, one of which is to induce systemic tolerance to vectored antibody. It is well established that the hepatic gene transfer can induce systemic immune tolerance to transgene products. Here, we attempted to use hepatotropic AAV serotypes and hepatocyte-specific promoter for liver-specific gene expression to induce systemic immune tolerance to muscle-expressed transgene products including intracellular β-Galactosidase (β-gal) or secreted Ovalbumin (OVA). First, we tested if the transgene specific CD8+ T cells could be reduced by using liver-specific promoter. C57BL/6 mice were intravenously delivered with rAAV9 vectors expressing β-gal from either ubiquitous chicken β-actin (CB) promoter or liver-specific human thyroxine binding globulin (TBG) promoter. When compared to CB promoter, TBG can achieve significantly higher β-gal expression with reduced β-gal specific CD8+ T cells. To compare the humoral immune responses against the secrete transgene products, AAV8 vectors expressing OVA from CB or TBG were intravenously injected to C57BL/6 mice. AAV8.TBG.OVA injected mice showed significant higher OVA expression than AAV8.CB.OVA injected mice did. Interestingly, while the mice received rAAV1CB.OVA intramuscularly triggered robust anti-OVA IgG1 response with low and transient OVA expression, such responses in the mice treated with intravenously delivered both AAV8 vectors were negligible accompanied with high levels of sustained OVA expression. In addition, the high frequency of regulatory T cells and CD19+CD5+CD1d+ regulatory B cells are detected in the spleens from the mice intravenously delivered both AAV8OVA vectors. Finally, we tested whether hepatic restricted OVA gene transfer by AAV8.TBG.OVA can indeed induce immune tolerance to subsequently and intramuscularly delivered AAV1.CB.OVA in C57BL/6 mice. Again, very low levels of transient OVA expression and high levels of anti-OVA IgG1 were detected in the animals treated with PBS followed by intramuscular AAV1.CB.OVA, whereas the intravenously delivered AAV8.TBG.OVA vectors facilitated the subsequently muscle-directed AAV1-encoded OVA expression without antibody production. Our data suggest that both regulatory T and B cells may play roles in systemic immune tolerance induced by liver-derived transgene expression from liver tropic AAV vector.

586. Development of Anti-Angiogenic miRNA Therapeutics for Corneal Neovascularization

GG and XX are co-corresponding authorsCorneal diseases are the second major cause of blindness worldwide. Corneal neovascularization (CNV) is one of the most common pathological processes in corneal diseases, leading to vision loss or even blindness in patients. However, even though different treatments for CNV are available in clinic, 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. Therefore, modulating expression of angiogenesis-related miRNAs might be an effective approach for treating this blindness-causing corneal lesion. To this end, we first tested four angiogenesis-related miRNAs (i. e. miR-126, miR-10, miR-17a and miR-92) for their role in angiogenesis in vitro by assessing cell proliferation and tube formation of VEGF-stimulated human umbilical vein endothelial cells (HUVECs) transfected with the plasmid constructs overexpressing the pri-miRNAs or Tough Decoy (Tud) RNAs inhibitory to the corresponding target miRNAs. We found that TuD-miR-10b, pri-miR-17a and pri-miR-92 were significantly inhibitory for the cell proliferation and tube formation of HUVECs, while TuD-miR-126-5p could impede HUVEC tube formation but not proliferation. Our data reveal that miR-126 and miR-10 are pro-angiogenic regulators, whereas miR-17a and miR-92 are negative regulators of angiogenesis, suggesting the feasibility of using miRNA as therapeutic targets to modulate angiogenesis in CNV. We next set out to identify more target miRNAs that play roles in CNV. To date, there has been no report on miRNA expression profile of CNV. Using Nanostring technologies and the classic alkali-burn induced CNV mouse model, we profiled small RNAs prepared from the corneal tissues harvested from the study mice before and days 5, 10 and 15 after alkali injury for expression levels of 618 mouse miRNAs. We found 35 up-regulated and 3 down-regulated miRNAs in the mouse neovascularized corneas. Currently, we are in the process of confirming expression profiles of those candidate target miRNAs by qRT-PCR. For proof-of-concept, we proposed to use rAAV vector for efficient delivery of those pri-miRNAs or their corresponding Tud RNAs to the corneas of alkali-burn induced CNV mice and evaluate their potency in preventing or treating CNV. To this end, we first assessed several serotypes of rAAV EGFP for gene transfer efficiency in mouse corneas after delivery by ectopic eye drops, or intrastromal or subconjunctival injections to identify the most efficient AAV serotype and method of delivery for miRNA therapeutics. The results of those experiments will be presented. In sum, our study has great clinical relevance; 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 clinical option for treating CNV.

Vector sequences are not detected in tumor tissue from research subjects with ornithine transcarbamylase deficiency who previously received adenovirus gene transfer.

A 66-year-old woman heterozygous for a mutation in the ornithine transcarbamylase gene (Otc) participated in a phase I gene therapy trial for OTC deficiency. She received an adenovirus (Ad) vector expressing the functional OTC gene by intraportal perfusion. Fourteen years later she developed and subsequently died of hepatocellular carcinoma. A second subject, a 45-year-old woman, enrolled in the same trial presented with colon cancer 15 years later. We sought to investigate a possible association between the development of a tumor and prior adenoviral gene transfer in these two subjects. We developed and validated a sensitive nested polymerase chain reaction assay for recovering recombinant Ad sequences from host tissues. Using this method, we could not detect any Ad vector DNA in either tumor or normal tissue from the two patients. Our results are informative in ruling out the possibility that the adenoviral vector might have contributed to the development of cancer in those two subjects.