Evaluation of Aav Capsids and Delivery Approaches for Hereditary Hemorrhagic Telangiectasia Gene Therapy.

Nosebleeds and intracranial hemorrhage from brain arteriovenous malformations (bAVMs) are among the most devastating symptoms of patients with hereditary hemorrhagic telangiectasis (HHT). All available managements have limitations. We showed that intravenous delivery of soluble FMS-related tyrosine kinase 1 using an adeno-associated viral vector (AAV9-sFLT1) reduced bAVM severity of endoglin deficient mice. However, minor liver inflammation and growth arrest in young mice were observed. To identify AAV variants and delivery methods that can best transduce brain and nasal tissue with an optimal transduction profile, we compared 3 engineered AAV capsids (AAV.cc47, AAV.cc84 and AAV1RX) with AAV9. A single-stranded CBA promoter driven tdTomato transgene was packaged in these capsids and delivered intravenously (i.v.) or intranasally (i.n.) to wild-type mice. A CMV promoter driven Alk1 transgene was packaged into AAV.cc84 and delivered to PdgfbiCre;Alk1 f/f mice through i.v. injection followed by brain AVM induction. Transduced cells in different organs, vessel density and abnormal vessels in the bAVMs, and liver inflammation were analyzed histologically. Liver and kidney function were measured enzymatically. Compared to other viral vectors, AAV.cc84, after i.v. delivery, transduced a high percentage of brain ECs and few hepatocytes; whereas after i.n. delivery, AAV.cc84 transduced ECs and perivascular cells in the brain, and ECs, epithelial cells, and skeletal muscles in the nose with minimum hepatocyte transduction. No changes to liver or kidney function were detected. Delivery of AAV.cc84-Alk1 through i.v. to PdgfbiCre;Alk1 f/f mice reduced bAVM severity. In summary, we propose that AAV.cc84-Alk1 is a promising candidate for developing gene therapy in HHT patients.

Evaluation of AAV Capsids and Delivery Approaches for Hereditary Hemorrhagic Telangiectasia Gene Therapy.

Nosebleeds and intracranial hemorrhage from brain arteriovenous malformations (bAVMs) are among the most devastating symptoms of patients with hereditary hemorrhagic telangiectasis (HHT). All available managements have limitations. We showed that intravenous (i.v.) delivery of soluble Feline McDonough Sarcoma (FMS)-related tyrosine kinase 1 using an adeno-associated viral vector (AAV9-sFLT1) reduced bAVM severity of endoglin deficient mice. However, minor liver inflammation and growth arrest in young mice were observed. To identify AAV variants and delivery methods that can best transduce brain and nasal tissue with an optimal transduction profile, we compared 3 engineered AAV capsids (AAV.cc47, AAV.cc84, and AAV1RX) with AAV9. A single-stranded CBA promoter driven tdTomato transgene was packaged in these capsids and delivered i.v. or intranasally (i.n.) to wild-type mice. A CMV promoter driven Alk1 transgene was packaged into AAV.cc84 and delivered to PdgfbiCre;Alk1f/f mice through i.v. followed by bAVM induction. Transduced cells in organs, vessel density, abnormal vessels in the bAVMs, and liver inflammation were analyzed histologically. Liver and kidney function were measured enzymatically. Compared to other viral vectors, AAV.cc84, after i.v. delivery, transduced a high percentage of brain endothelial cells (ECs) and few hepatocytes; whereas after i.n. delivery, AAV.cc84 transduced ECs and perivascular cells in the brain, and ECs, epithelial cells, and muscles in the nose with minimum hepatocyte transduction. No changes to liver or kidney function were detected. The delivery of AAV.cc84-Alk1 through i.v. to PdgfbiCre;Alk1f/f mice reduced bAVM severity. In summary, we propose that AAV.cc84-Alk1 is a promising candidate for developing gene therapy in HHT patients.

Capsid-mediated control of adeno-associated viral transcription determines host range.

Adeno-associated virus (AAV) is a member of the genus Dependoparvovirus, which infects a wide range of vertebrate species. Here, we observe that, unlike most primate AAV isolates, avian AAV is transcriptionally silenced in human cells. By swapping the VP1 N terminus from primate AAVs (e.g., AAV8) onto non-mammalian isolates (e.g., avian AAV), we identify a minimal component of the AAV capsid that controls viral transcription and unlocks robust transduction in both human cells and mouse tissue. This effect is accompanied by increased AAV genome chromatin accessibility and altered histone methylation. Proximity ligation analysis reveals that host factors are selectively recruited by the VP1 N terminus of AAV8 but not avian AAV. Notably, these include AAV essential factors implicated in the nuclear factor κB pathway, chromatin condensation, and histone methylation. We postulate that the AAV capsid has evolved mechanisms to recruit host factors to its genome, allowing transcriptional activation in a species-specific manner.

A humanized mouse model for adeno-associated viral gene therapy.

Clinical translation of AAV-mediated gene therapy requires preclinical development across different experimental models, often confounded by variable transduction efficiency. Here, we describe a human liver chimeric transgene-free Il2rg-/-/Rag2-/-/Fah-/-/Aavr-/- (TIRFA) mouse model overcoming this translational roadblock, by combining liver humanization with AAV receptor (AAVR) ablation, rendering murine cells impermissive to AAV transduction. Using human liver chimeric TIRFA mice, we demonstrate increased transduction of clinically used AAV serotypes in primary human hepatocytes compared to humanized mice with wild-type AAVR. Further, we demonstrate AAV transduction in human teratoma-derived primary cells and liver cancer tissue, displaying the versatility of the humanized TIRFA mouse. From a mechanistic perspective, our results support the notion that AAVR functions as both an entry receptor and an intracellular receptor essential for transduction. The TIRFA mouse should allow prediction of AAV gene transfer efficiency and the study of AAV vector biology in a preclinical human setting.

Engineering Synthetic circRNAs for Efficient CNS Expression.

Circular RNAs (circRNAs) have recently emerged as a promising modality for gene and RNA-based therapies. They are more stable than their linear counterpart and can be designed for efficient expression in different cell and tissue types. In this chapter, we developed different backsplicing circRNA cassettes that can enable efficient gene expression in various cell and tissue types. Furthermore, we packaged cassettes encoding circRNAs into adeno-associated viral (AAV) vectors that can be delivered via intracerebroventricular (ICV) injections to achieve expression in murine brain tissue. We provide detailed methods for the design of backsplicing circRNAs, circRNA detection, and generation of AAV-circRNA vectors for CNS dosing and expression in mice.

Gene delivery followed by ex vivo lung perfusion using an adeno-associated viral vector in a rodent lung transplant model.

OBJECTIVE: Ex vivo lung perfusion has emerged as a platform for organ preservation, evaluation, and restoration. Gene delivery using a clinically relevant adeno-associated vector during ex vivo lung perfusion may be useful in optimizing donor allografts while the graft is maintained physiologically active. We evaluated the feasibility of adeno-associated vector-mediated gene delivery during ex vivo lung perfusion in a rat transplant model. Additionally, we assessed off-target effects and explored different routes of delivery. METHODS: Rat heart-lung blocks were procured and underwent 1-hour ex vivo lung perfusion. Before ex vivo lung perfusion, 4e11 viral genome luciferase encoding adeno-associated vector 9 was administered via the left bronchus (Br group, n = 4), via the left pulmonary artery (PA group, n = 3), or directly into the circuit (Circuit group, n = 3). Donor lungs in the Control group (n = 3) underwent ex vivo lung perfusion without adeno-associated vector 9. Only the left lung was transplanted. Animals underwent bioluminescence imaging weekly before being killed at 2 weeks. Tissues were collected for luciferase activity measurement. RESULTS: All recipients tolerated the transplant well. At 2 weeks post-transplant, luciferase activity in the transplanted lung was significantly higher among animals in the Br group compared with the other 3 groups (Br: 1.1 × 106 RLU/g, PA: 8.3 × 104 RLU/g, Circuit: 3.8 × 103 RLU/g, Control: 2.5 × 103 RLU/g, P = .0003). No off-target transgene expression was observed. CONCLUSIONS: In this work, we demonstrate that a clinically relevant adeno-associated vector 9 vector mediates gene transduction during ex vivo lung perfusion in rat lung grafts when administered via the airway and potentially the pulmonary artery. Our preliminary results suggest a higher transduction efficiency when adeno-associated vector 9 was delivered via the airway, and delivery during ex vivo lung perfusion reduces off-target effects after graft implant.

Structure-guided AAV capsid evolution strategies for enhanced CNS gene delivery.

Over the past 5 years, our laboratory has systematically developed a structure-guided library approach to evolve new adeno-associated virus (AAV) capsids with altered tissue tropism, higher transduction efficiency and the ability to evade pre-existing humoral immunity. Here, we provide a detailed protocol describing two distinct evolution strategies using structurally divergent AAV serotypes as templates, exemplified by improving CNS gene transfer efficiency in vivo. We outline four major components of our strategy: (i) structure-guided design of AAV capsid libraries, (ii) AAV library production, (iii) library cycling in single versus multiple animal models, followed by (iv) evaluation of lead AAV vector candidates in vivo. The protocol spans ~95 d, excluding gene expression analysis in vivo, and can vary depending on user experience, resources and experimental design. A distinguishing attribute of the current protocol is the focus on providing biomedical researchers with 3D structural information to guide evolution of precise 'hotspots' on AAV capsids. Furthermore, the protocol outlines two distinct methods for AAV library evolution consisting of adenovirus-enabled infectious cycling in a single species and noninfectious cycling in a cross-species manner. Notably, our workflow can be seamlessly merged with other RNA transcript-based library strategies and tailored for tissue-specific capsid selection. Overall, the procedures outlined herein can be adapted to expand the AAV vector toolkit for genetic manipulation of animal models and development of human gene therapies.

Spinal cord repair is modulated by the neurogenic factor Hb-egf under direction of a regeneration-associated enhancer.

Unlike adult mammals, zebrafish regenerate spinal cord tissue and recover locomotor ability after a paralyzing injury. Here, we find that ependymal cells in zebrafish spinal cords produce the neurogenic factor Hb-egfa upon transection injury. Animals with hb-egfa mutations display defective swim capacity, axon crossing, and tissue bridging after spinal cord transection, associated with disrupted indicators of neuron production. Local recombinant human HB-EGF delivery alters ependymal cell cycling and tissue bridging, enhancing functional regeneration. Epigenetic profiling reveals a tissue regeneration enhancer element (TREE) linked to hb-egfa that directs gene expression in spinal cord injuries. Systemically delivered recombinant AAVs containing this zebrafish TREE target gene expression to crush injuries of neonatal, but not adult, murine spinal cords. Moreover, enhancer-based HB-EGF delivery by AAV administration improves axon densities after crush injury in neonatal cords. Our results identify Hb-egf as a neurogenic factor necessary for innate spinal cord regeneration and suggest strategies to improve spinal cord repair in mammals.

Rescue of glutaric aciduria type I in mice by liver-directed therapies.

Glutaric aciduria type I (GA-1) is an inborn error of metabolism with a severe neurological phenotype caused by the deficiency of glutaryl-coenzyme A dehydrogenase (GCDH), the last enzyme of lysine catabolism. Current literature suggests that toxic catabolites in the brain are produced locally and do not cross the blood-brain barrier. In a series of experiments using knockout mice of the lysine catabolic pathway and liver cell transplantation, we uncovered that toxic GA-1 catabolites in the brain originated from the liver. Moreover, the characteristic brain and lethal phenotype of the GA-1 mouse model was rescued by two different liver-directed gene therapy approaches: Using an adeno-associated virus, we replaced the defective Gcdh gene or we prevented flux through the lysine degradation pathway by CRISPR deletion of the aminoadipate-semialdehyde synthase (Aass) gene. Our findings question the current pathophysiological understanding of GA-1 and reveal a targeted therapy for this devastating disorder.

AAV9-mediated gene delivery to liver grafts during static cold storage in a rat liver transplant model.

Introduction: Recombinant adeno-associated virus (rAAV) is a novel strategy used clinically for gene delivery, but has not been characterized in the context of organ transplantation. We sought to determine the efficacy of rAAV-mediated gene delivery during static cold storage (SCS) prior to liver transplantation. Methods: A triple-plasmid transfection protocol was used to produce rAAV subtype-9 vectors containing firefly luciferase genomes in HEK293 cells. Lewis rat liver grafts were flushed and stored in cold HTK solution. Three experimental groups received rAAV at different doses, administered via the portal vein as a bolus during SCS. A control group did not receive rAAV (N = 2). Recipients then underwent syngeneic liver transplantation. Bioluminescence imaging to quantify in vivo luciferase expression was performed on post-operative days 7, 14, 28, and 56. Results: Control animals demonstrated no bioluminescent activity, while animals receiving rAAV-treated livers had increasing bioluminescence, peaking at four weeks but sustained to the eight-week endpoint. This result was confirmed by experimental endpoint tissue luciferase activity assay. Discussion: rAAV mediates gene transduction in liver grafts when administered during SCS and has potential for gene therapy applications in solid organ transplantation.

Adeno-associated virus mediates gene transduction after static cold storage treatment in rodent lung transplantation.

OBJECTIVE: Adeno-associated virus is a clinically used gene therapy vector but has not been studied in lung transplantation. We sought to determine the efficacy of adeno-associated virus delivery during static cold storage via the airway versus the pulmonary artery before lung transplantation in a rodent model. METHODS: Lewis rat lung grafts were treated with a dose of 8e8 or 4e9 viral genome/µL recombinant adeno-associated virus subtype-9 vectors containing firefly luciferase genomes administered via the pulmonary artery or airway during cold storage. A control group did not receive adeno-associated virus. Recipient syngeneic rats then underwent single left lung transplantation. Animals underwent bioluminescence imaging on postoperative days 7, 14, 28, and 56. Explanted tissues were prepared as lysates to quantify luciferase activity. Immunohistochemistry was performed to evaluate cellular transgene expression patterns. RESULTS: Control animals with no luminescent signal produced a background radiance of 6.1e4 p/s/cm2/sr. In the airway delivery group, mean radiance was greater than the control at 4e9 viral genome/µL postoperative day 7 radiance 6.9e4 p/s/cm2/sr (P = .04). In the pulmonary artery delivery group, we observed greater in vivo luminescence in animals receiving 4e9 viral genome/µL compared with all other groups. However, analysis of tissue lysate revealed greater luminescence in the airway delivery group and suggested off-target expression in heart and liver tissue in the pulmonary artery delivery group. Immunohistochemistry demonstrated transgene staining in distal airway epithelium and alveoli but sparing of the vasculature in the airway delivery group. CONCLUSIONS: Adeno-associated virus mediates gene transduction during static cold storage in rat lung isografts when administered via the airway and pulmonary artery. Airway administration leads to robust transgene expression in respiratory epithelial cells, whereas pulmonary artery administration targets alternative cell types and increases extrapulmonary transgene expression.

Cross-species evolution of a highly potent AAV variant for therapeutic gene transfer and genome editing.

Recombinant adeno-associated viral (AAV) vectors are a promising gene delivery platform, but ongoing clinical trials continue to highlight a relatively narrow therapeutic window. Effective clinical translation is confounded, at least in part, by differences in AAV biology across animal species. Here, we tackle this challenge by sequentially evolving AAV capsid libraries in mice, pigs and macaques. We discover a highly potent, cross-species compatible variant (AAV.cc47) that shows improved attributes benchmarked against AAV serotype 9 as evidenced by robust reporter and therapeutic gene expression, Cre recombination and CRISPR genome editing in normal and diseased mouse models. Enhanced transduction efficiency of AAV.cc47 vectors is further corroborated in macaques and pigs, providing a strong rationale for potential clinical translation into human gene therapies. We envision that ccAAV vectors may not only improve predictive modeling in preclinical studies, but also clinical translatability by broadening the therapeutic window of AAV based gene therapies.

Global assessment of organ specific basal gene expression over a diurnal cycle with analyses of gene copies exhibiting cyclic expression patterns.

BACKGROUND: Studying functional divergences between paralogs that originated from genome duplication is a significant topic in investigating molecular evolution. Genes that exhibit basal level cyclic expression patterns including circadian and light responsive genes are important physiological regulators. Temporal shifts in basal gene expression patterns are important factors to be considered when studying genetic functions. However, adequate efforts have not been applied to studying basal gene expression variation on a global scale to establish transcriptional activity baselines for each organ. Furthermore, the investigation of cyclic expression pattern comparisons between genome duplication created paralogs, and potential functional divergence between them has been neglected. To address these questions, we utilized a teleost fish species, Xiphophorus maculatus, and profiled gene expression within 9 organs at 3-h intervals throughout a 24-h diurnal period. RESULTS: Our results showed 1.3-21.9% of genes in different organs exhibited cyclic expression patterns, with eye showing the highest fraction of cycling genes while gonads yielded the lowest. A majority of the duplicated gene pairs exhibited divergences in their basal level expression patterns wherein only one paralog exhibited an oscillating expression pattern, or both paralogs exhibit oscillating expression patterns, but each gene duplicate showed a different peak expression time, and/or in different organs. CONCLUSIONS: These observations suggest cyclic genes experienced significant sub-, neo-, or non-functionalization following the teleost genome duplication event. In addition, we developed a customized, web-accessible, gene expression browser to facilitate data mining and data visualization for the scientific community.

Defining Transcription Regulatory Elements in the Human Frataxin Gene: Implications for Gene Therapy.

Friedreich's ataxia (FRDA) is the most common inherited form of ataxia in humans. It is caused by severe downregulation of frataxin (FXN) expression instigated by hyperexpansion of the GAA repeats located in intron 1 of the FXN gene. Despite numerous studies focused on identifying compounds capable of stimulating FXN expression, current knowledge regarding cis-regulatory elements involved in FXN gene expression is lacking. Using a combination of episomal and genome-integrated constructs, we defined a minimal endogenous promoter sequence required to efficiently drive FXN expression in human cells. We generated 19 constructs varying in length of the DNA sequences upstream and downstream of the ATG start codon. Using transient transfection, we evaluated the capability of these constructs to drive FXN expression. These analyses allowed us to identify a region of the gene indispensable for FXN expression. Subsequently, selected constructs containing the FXN expression control regions of varying lengths were site specifically integrated into the genome of HEK293T and human-induced pluripotent stem cells (iPSCs). FXN expression was detected in iPSCs and persisted after differentiation to neuronal and cardiac cells, indicating lineage independent function of defined regulatory DNA sequences. Finally, based on these results, we generated AAV encoding miniFXN genes and demonstrated in vivo FXN expression in mice. Results of these studies identified FXN sequences necessary to express FXN in human and mouse cells and provided rationale for potential use of endogenous FXN sequence in gene therapy strategies for FRDA.

Fluorescent light exposure incites acute and prolonged immune responses in zebrafish (Danio rerio) skin.

Artificial light produces an emission spectrum that is considerably different than the solar spectrum. Artificial light has been shown to affect various behavior and physiological processes in vertebrates. However, there exists a paucity of data regarding the molecular genetic effects of artificial light exposure. Previous studies showed that one of the commonly used fluorescent light source (FL; 4100K or "cool white") can affect signaling pathways related to maintenance of circadian rhythm, cell cycle progression, chromosome segregation, and DNA repair/recombination in the skin of male Xiphophorus maculatus. These observations raise questions concerning the kinetics of the FL induced gene expression response, and which biological functions become modulated at various times after light exposure. To address these questions, we exposed zebrafish to 4100K FL and utilized RNA-Seq to assess gene expression changes in skin at various times (1 to 12h) after FL exposure. We found 4100K FL incites a robust early (1-2h) transcriptional response, followed by a more protracted late response (i.e., 4-12h). The early transcriptional response involves genes associated with cell migration/infiltration and cell proliferation as part of an overall increase in immune function and inflammation. The protracted late transcriptional response occurs within gene sets predicted to maintain and perpetuate the inflammatory response, as well as suppression of lipid, xenobiotic, and melatonin metabolism.

Characterization of basal gene expression trends over a diurnal cycle in Xiphophorus maculatus skin, brain and liver.

Evolutionarily conserved diurnal circadian mechanisms maintain oscillating patterns of gene expression based on the day-night cycle. Xiphophorus fish have been used to evaluate transcriptional responses after exposure to various light sources and it was determined that each source incites distinct genetic responses in skin tissue. However, basal expression levels of genes that show oscillating expression patterns in day-night cycle, may affect the outcomes of such experiments, since basal gene expression levels at each point in the circadian path may influence the profile of identified light responsive genes. Lack of knowledge regarding diurnal fluctuations in basal gene expression patterns may confound the understanding of genetic responses to external stimuli (e.g., light) since the dynamic nature of gene expression implies animals subjected to stimuli at different times may be at very different stages within the continuum of genetic homeostasis. We assessed basal gene expression changes over a 24-hour period in 200 select Xiphophorus gene targets known to transcriptionally respond to various types of light exposure. We identified 22 genes in skin, 36 genes in brain and 28 genes in liver that exhibit basal oscillation of expression patterns. These genes, including known circadian regulators, produced the expected expression patterns over a 24-hour cycle when compared to circadian regulatory genes identified in other species, especially human and other vertebrate animal models. Our results suggest the regulatory network governing diurnal oscillating gene expression is similar between Xiphophorus and other vertebrates for the three Xiphophorus organs tested. In addition, we were able to categorize light responsive gene sets in Xiphophorus that do, and do not, exhibit circadian based oscillating expression patterns.