Thesis advisor: Eranthie Weerapana / Adeno-Associated Virus (AAV) has been developed into a powerful therapeutic tool - in the last ten years it has acted as a gene-delivery vehicle in several approved therapeutics and many more therapeutics on trial. Despite extensive research, gaps in our understanding of AAV’s infectious cycle still exist, and further development is needed for the creation of improved gene therapy vectors. Technology to incorporate Unnatural Amino Acids (UAAs) into the AAV capsid has recently been developed, and could aid in both furthering our understanding of AAV’s biology and in the therapeutic advancement of AAV. In this work, we demonstrate how the functionalization of the AAV capsid using UAA incorporation can advance our control over the AAV capsid and aid in probing and manipulating AAV biology. We describe our use UAA incorporation to place a bio-orthogonal reactive handle into AAV’s capsid followed by functionalization with a targeting moiety and demonstrate the unprecedented amount of control that UAA incorporation provides in the creation of a functional virus conjugate. We are able to control both the precise placement and the stoichiometry of the targeting moiety on the AAV capsid, providing a platform that, for the first time, can undergo rigorous optimization analogous to that which medicinal chemists put small molecules through. We also describe the creation of a new platform to site-specifically modify the AAV capsid using cysteine incorporation, a technique that retains the ability to site-specifically modify the capsid as UAA incorporation does, but does not require the excess machinery that UAA incorporation requires. Next we discuss the incorporation of a photocaging amino acid, NBK, into the AAV capsid. Using NBK, we were able to effectively block AAV’s primary binding interaction with Heparan Sulfate Proteoglycan (HSPG) and control the timing of AAV infection using light to chemically remove the photo-protecting group. While photocaging the HSPG interaction is only a proof of concept, it demonstrates the remarkable amount of control that UAA incorporation affords, and lends insight to what could be accomplished using the functionalities that can be placed on the AAV capsid with UAAs. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

A novel strategy for site-specific DNA transformation of plants has been proposed and the first component of the system developed. The proposed method overcomes the limitations of current techniques by providing a specific integration site for the insertion of transgenes using features of the adeno-associated virus (AAV) life cycle. In the absence of helper virus, AAV integrates into a specific location on human chromosome 19, the AAVS1 locus. The sequence for AAV integration was introduced into the model plant Arabidopsis thaliana using Agrobacterium tumefaciens-mediated transformation. A portion of the human AAVS1 sequence, including the Rep binding site (RBS) and terminal resolution site (TRS), was cloned between T-DNA borders of the Agrobacterium Ti plasmid. The reporter gene, b-glucuronidase (GUS) was inserted proximal to AAVS1 in the plasmid for use in screening for the presence of T-DNA. In addition, it will serve as an indicator of the expression level expected for transgene inserted into AAVS1 by recombinant AAV. PCR amplification, dideoxy sequencing, GUS expression assays and genomic Southern blots were performed to examine putative transgenic plants for the presence of the AAVS1 sequence. / Master of Science

Gene therapy has emerged as a revolutionary approach for treating genetic disorders, holding great promise for improving patient outcomes. Among the various viral vectors used for delivery of therapeutic transgenes, Adeno-Associated Viruses (AAVs) have gained prominence due to their favorable characteristics including low immunogenicity, long-term gene expression, and the ability to target both dividing and non-dividing cells. However, AAV’s are associated with the high costs of production and challenges with production of a high-quality virus, limiting AAV’s utilization and widespread use. In this study, we aimed to develop a high-throughput screening assay targeting AAV production enhancers, thus addressing the manufacturing obstacles and advancing the affordability and accessibility of gene therapies. To help overcome the limitations and expenses associated with AAV manufacturing, an innovative high-throughput screening assay was developed with the intent to identify cell culture additives/conditions which maximize AAV production. We optimized various parameters, including the transgene, producer and reporter cell lines, harvest timings and methods, and transduction techniques. The optimized screening assay was employed to evaluate novel compounds across several timings of addition, for their ability to enhance AAV production. Notably, several compounds indicated transfection enhancing capabilities up to 3.4-fold and the developed assays final variability was below 14%. Additionally, compound combinations were assessed to uncover potential additive and synergistic effects that could further enhance AAV productivity. In conclusion, our study presents a significant advancement in targeting the manufacturing challenges associated with AAV. By utilizing an optimized high-throughput screening assay, researchers and manufacturers can identify compounds that enhance AAV production, paving the way for cost-effective and scalable manufacturing processes. Ultimately, this progress holds the potential to improve the affordability, accessibility, and impact of gene therapies for patients worldwide.

Thesis advisor: Abhishek Chatterjee / Directed evolution is a powerful technique to expand chemical space in biological systems. In particular, this method has been used to develop cellular machinery to enable genetic code expansion (GCE), the incorporation of unnatural amino acids (UAAs) into proteins during the translation process. GCE relies on evolving an aminoacyl tRNA synthetase (aaRS) and tRNA pair from a different domain of life to incorporate a UAA into proteins in their new host, as these evolutionarily distant pairs are less likely to be cross-reactive with host pairs. The aaRS and tRNA must meet a number of conditions to be useful for GCE: the pair must be orthogonal (non-cross-reactive) to the host’s native aaRS/tRNA pairs in order to ensure site-specific UAA incorporation; the aaRS must have an active site suited to accept the shape of the UAA; and the tRNA must cooperate with the host ribosome, elongation and release factors, and other translational machinery to efficiently incorporate the UAA into the protein. Numerous aaRS/tRNA pairs have been evolved to allow incorporation of diverse UAAs in bacteria due to the tractable nature of these organisms for directed evolution experiments. While an aaRS evolved in bacteria to charge a novel UAA can be used in eukaryotes, tRNAs cannot be evolved for GCE in bacteria and then used in eukaryotes because they will not have evolved in the presence of the correct translational machinery. It is necessary to evolve tRNAs directly in their host cells. Unfortunately for researchers working on GCE in mammalian cells, it is difficult to perform directed evolution on small gene products in these hosts. Transformation efficiency in mammalian cells is poor, and transient transfection yields heterogeneous DNA distribution to target cells, making selection based on performance of individual library members impossible. Viruses are an ideal DNA delivery vector for mammalian cells, as production of recombinant viruses allows control over library member generation, and viruses can be delivered with exquisite copy number control. The Chatterjee lab recently developed a platform, Virus-Assisted Directed Evolution of tRNAs (VADER), using adeno-associated virus (AAV) to evolve tRNAs for GCE directly in mammalian cells. While VADER is the first directed evolution platform that allows the evolution of small gene products in mammalian cells, its efficiency is limited by its continued reliance on transient transfection to deliver non-library DNA that is necessary for the production of rAAV. To overcome this limitation, baculovirus delivery vectors were developed to boost DNA delivery and AAV capsid production to improve virus production efficiency during selections. VADER allows the evolution of tRNAs to incorporate certain UAAs, but the technique relies on installing a UAA into the AAV capsid, which is sensitive to disruption caused by slight modifications in structure. To expand the scope of VADER to evolve tRNAs for UAAs that cannot be incorporated into the AAV capsid, an alternate selection handle (Assembly Activating Protein, or AAP) was deleted from the genome and provided in trans to incorporate 5-hydroxytryptophan (5HTP). Incorporating the UAA into this flexible protein allows UAA-dependent production of AAV and expands the scope of tRNAs that can be evolved in mammalian cells. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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No description available.

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease caused by mutations in a single gene, the cystic fibrosis transmembrane conductance regulator (CFTR). CF affects multiple organ systems, but the major cause of morbidity and mortality is due to disease in the lungs. In theory, using gene therapy to deliver a correct copy of CFTR to the cells of the airway epithelium could result in a lifelong cure. Adeno-associated virus (AAV) is a single stranded DNA virus that is a promising candidate vector for gene therapy of multiple diseases, and numerous clinical trials are currently underway. Despite recent clinical successes, several challenges still impede wider application of AAV gene therapy to numerous diseases, including CF, as AAV-mediated gene transfer to the airways remains below the level needed for therapeutic efficacy for CF. We hypothesized that the low transduction efficiency of AAV in the airways could be overcome by using directed evolution of AAV in organotypic human and pig airway models, and in vivo in the lungs of pigs to select novel AAV capsid variants with improved infectious properties. We discovered a highly infectious, novel AAV that was a chimera of AAV2 and AAV5 with one point mutation (A581T) which we called AAV2.5T. We found that AAV2.5T mediated gene transfer significantly better than its parental serotypes, and corrected the chloride transport defect in CF human airway epithelial cultures. We determined that AAV2.5T developed increased binding to the apical surface of human airway epithelial cells, and that it has evolved to utilize specific 2,3N-linked sialic acid residues on the cell surface that mediate rapid internalization and subsequent infection. Thus, sialic acid serves as not just an attachment factor but is also required for AAV2.5T internalization, possibly representing an important rate-limiting step for other viruses that use sialic acids. Additionally, we utilized directed evolution in vivo in the lungs of pigs to select a novel AAV capsid that is identical to AAV2 except for five point mutations, which we called AAV2H22. We found that AAV2H22 mediated gene transfer to pig airway epithelial cultures significantly better than AAV2, and that it had evolved altered receptor binding. We also found that directed evolution in vitro in human and pig airway epithelial cultures results in the selection of distinct viruses for the two species, and that maintaining different selection stringencies results in the recovery of different AAV variants. Finally, we utilized Hoechst 33342, a DNA binding compound which was previously found to increase AAV transduction in cell lines, to increase AAV-mediated gene expression in primary human airway epithelia. We determined that the mechanism of this effect was due to activation of the CMV promoter. The findings from this research have significant implications for our understanding of AAV biology and for pulmonary gene therapy.

Recombinant adeno-associated virus (rAAV) is a widely used gene therapy vector. Although a wide range of rAAV serotypes can effectively enter most cell types, their transduction efficiencies (i.e., transgene expression) can vary widely depending on the target cell type. Integrins play important roles as co-receptors for rAAV infection, however, it remains unclear how integrin-dependent and -independent mechanisms of rAAV endocytosis influence the efficiency of intracellular virus processing and ultimately transgene expression. In this thesis, I examined the contribution of integrin-mediated endocytosis to transduction of fibroblasts by rAAV2. I found that promoting AAV2/integrin binding with Mn++ greatly enhanced (~17-fold) rAAV2 transduction independently of cell binding and endocytosis. Subcellular localization studies of rAAV2 demonstrated that integrin activation by Mn++ promoted AAV2 aggregation on alpha5 and beta1 integrins and recruitment of the cytosolic integrin effector protein vinculin. Focal adhesion kinase (FAK), a down stream effector of integrin signals, was essential for AAV/integrin complex endocytosis and transduction, but not AAV2 recruitment to integrins. Recruitment of FAK to AAV2/integrin complexes was increased by transiently trapping the endocytic event at the plasma membrane by pharmacologic inhibition of dynasore. This also increased the size of AAV2 clusters found beneath the cell at FAK/integrin complexes resembling immature filopodia and caused a large, FAK-dependent (75-fold) increase in AAV2 transduction. These findings support a model whereby integrin activation at the cell surface can redirect rAAV2 toward a FAK-dependent entry pathway that is more productive for cellular transduction. This pathway appears to be conserved for other rAAV serotypes that contain a capsid integrin-binding domain (AAV1 and 6).

Age-related macular degeneration (AMD) is the leading cause for visual impairment and blindness in the elder population of developed countries. The primary underlying cause for significant visual loss is the choroidal neovascularization (CNV). Current treatment strategies for AMD include laser photocoagulation, photodynamic therapy (PDT) and excision of neovascular membranes, but have met with limited success. In our previous studies, we demonstrated that gene delivery of angiogenesis inhibitor vasostatin (VS) attenuated the corneal neovascularization in animals. The primary objective of this study was to investigate gene delivery of vasostatin (VS) attenuated the choroidal neovascularization in animals. Retinal and visual function will be evaluated. However, systematic expression of angiogenesis inhibitor may bring adverse effects to physiological processes. The feasibility, efficiency and safety of gene delivery with systemic and local routes were evaluated. Intramuscular polymer-based gene delivery had no side effect such as virus vector and revealed the safety. Recombinant adenovirus (Ad) was used gene delivery system because of its high titer, wide host range, and transduction efficiency. Adeno-associated virus (AAV) represents highly efficient that can facilirate long-term transduction. We propose to improve the efficacy and safety of VS gene delivery, and to search for the effective delivery route and other adjuvant therapy in conjunction with VS for treatment of CNV. Recently, PDT with veteporfin is an established treatment for subfoveal CNV secondary to AMD. We tried to compare the effect and safety of standard and reduced-dose light application PDT in an animal mocel of CNV. The 180-residue VS and its 48-residue (VS48) inhibited the migration and tube formation in cultured endothelial cells. Topical VS application suppresses the progression of laser-induced CNV via angiogenesis ihhibition, as well as in VS48. VS-48 inhibited the growthof vessels in arota rings. Electroretinograms (ERG) analysis revealed that topical VS-48 application for 21 days had no effect on rat retinal functions. Topical VS-48 treatment significantly reversed the CNV-induced alterations in ERG. Transfection of pCMV3-VS into muscle cells resulted in increased production and release of exogenous VS, which specifically inhibited the proliferation of endothelial cells. Rats treated with intrmuscular injection with PVP-VS also showed a significant reduction in the CNV lesions for at least 42 days. Subconjunctival injection with Ad vector revealed no retinal toxicity in ERG. Ad-luciferase via subconjunctival injections showed ocular expression for as long as 112 days by using bioluminescence image analysis in rodent. AAV-luciferase via subconjunctival injections showed ocular expression for as long as 365 days by using bioluminescence image analysis in mice, and AAV serotype 5-luciferase even showed expression lasting for 2 years. Suppression of laser photocoagulation¡Vinduced CNV by Ad-VS was documented in rat model. Combination therapies are important as treatment options. We demonstrated that PDT could effectively attenuate CNV in a rat model, and reduced doses, worked just as well as the standard dose. In the preliminary study of PDT combined topical VS application, treatment led to CNV attenuation more than alone with PDT. The above experiments would enable us to demonstrate that the vasostatin delivery might be a promising strategy for the treatment of AMD and other retinal or ocular disorders. Furthermore, the results from animal studies might be extrapolated for future clinical application.

Rhodopsin-linked retinitis pigmentosa (RP) is the most common form of autosomal dominant RP, an inherited retinal degeneration, in which rod degeneration is followed by secondary cone loss leading to loss of vision and blindness. The overall objective of this work was to develop an optimized gene replacement therapy, delivering the rhodopsin gene for rhodopsin- linked RP and establish whether secondary cone loss can be delayed. A fast-acting single mutant serotype 8 self-complementary adeno-associated virus vector was produced containing the human rhodopsin promoter and the human rhodopsin coding sequence. In vivo studies in rhodopsin knockout mouse showed that the vector administration led to widespread and robust expression of the transgene. Subretinal injection of the vector into three-week pups of rhodopsin knockout mice with cones expressing green fluorescent protein showed restoration of rod-derived electroretinogram (ERG) responses, and preservation of cone- driven ERG responses three months after injection. Similarly, the longitudinal follow-up with confocal scanning ophthalmoscopy found preservation of fluorescent cones up to three months after injection. Overall, these data provided evidence that the designed vector resulted in significant benefit to rod photoreceptors as well as in delay in secondary cone degeneration and built a basis for future use of this vector on dominant models of RP.