Browsing by Subject "AAV"
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Item Engineering Adeno-Associated Virus for Receptor-Mediated Gene Delivery(2021-03) Zdechlik, AlinaAdeno-associated virus (AAV) is a useful gene delivery tool for clinical and basic research applications. Since its discovery nearly 60 years ago, it has become a popular vector because of its small size and low immunogenicity. However, natural tissue tropisms are limited and frequently not useful. Past work modifying the capsid has been limited by structural constraints and a lack of modularity. I aimed to address this problem by creating a modular retargeting system in which the AAV capsid can be rapidly retargeted to any given antigen without newly mutating capsid proteins. I characterized AAV-antibody composites produced by incorporating a small DNA binding domain into one of the AAV capsid proteins and using chemical conjugation to attach the paired DNA sequence to an antibody. I demonstrated that these antibody-AAV conjugates are capable of infecting cells via the antibody-antigen interaction in immortalized and primary cells. Additionally, I created six capsid variants incorporating small targeting scaffolds into each AAV capsid protein. These variants will enable future researchers to select variants from scaffold libraries in vivo, making more specific, better targeting moieties. Using these retargeted vectors, I worked with colleagues to target prostate tumors in vivo and to deliver new payloads with therapeutic potential. A collaborator had recently developed an antibody against a prostate tumor stromal marker, fibroblast activation protein alpha (FAP). By conjugating this antibody to my modified virus, we were able to deliver a fluorescent marker specifically to the tumor. Next, we plan to deliver therapeutic payloads to treat the tumor with minimal off-target effects. Additionally, I assisted in designing and testing a version of prime editor suitable for delivery by AAV. While the efficiency of this tool is still relatively low, it represents an important starting point for adaption of prime editor for use in vivo. In future work, we hope to use antibody-targeted AAV to deliver this and other editing reagents in murine disease models. Finally, to further investigate the landscape of the AAV capsid, we prepared a domain insertion library using a protocol recently developed by the lab. The results from this study will provide valuable information about the plasticity of the AAV capsid that can be used by future researchers to create new variants with added functionality.Item Genome engineering in large animals for agricultural and biomedical applications(2013-08) Tan, WenfangPrecision genetics will enhance genome-based improvement of livestock for agriculture and biomedicine. This thesis aimed to modify large animal genomes with precision; as the technologies progressed, our capability expanded from random insertional transgenesis to nucleotide-level precision. It began with Sleeping Beauty (SB) transposon mediated rapid integration of dominant negative Myostatin alleles. All piglets generated from treated cells harbored the transgenes; however, we were unable to study phenotypes due to death of the founder animals. We then sought to introgress a SNP into porcine Myostatin through recombinant Adeno-associated Virus (rAAV) mediated gene targeting. We achieved a 2x10-4 targeting frequency but only one-half of the targeted colonies harbored the SNP. Similarly, we succeeded in porcine LDLR gene knockout; however, targeted clones were often confounded by "bystander" cells with only random insertions of the targeting vector. We turned to develop TALENs for efficient targeting of important genes. TALENs demonstrated high activity in both cultured primary fibroblasts and early stage embryos. A simple SB transposon based co-selection strategy enabled enrichment for TALEN modified cells and efficient isolation of modified clones: single gene mono- and bi-allelic modification was induced in up to 54% and 17% of colonies respectively. It also enabled isolation of colonies harboring large chromosomal deletions (10% of colonies) and inversions (4%) after treatment with two TALEN pairs. We derived miniature swine models of familial hypercholesterolemia from LDLR mono- and bi-allelic TALEN-knockout fibroblasts. We next utilized TALEN and CRISPR/Cas9 stimulated homology-directed repair (HDR) to edit genes with oligonucleotide, plasmid, and rAAV templates without any drug selection. We first introgressed a bovine POLLED allele into horned dairy bull fibroblasts to circumvent manual dehorning. We also introduced single-nucleotide alterations or small indels into 14 additional genes in pig, cattle and sheep, into 10-50% of cells from fibroblast populations treated with TALEN mRNA and oligonucleotides. Up to 67% of propagated colonies harbored the intended edits and over one-half were homozygous. Some edits were naturally occurring SNP alleles, equivalent to non-meiotic inter- or intra-species introgression of valuable alleles. We created pig models for infertility and colon cancer from colonies with TALEN-HDR knockout alleles in DAZL and APC.Item Molecular therapy for mucopolysaccharidosis Type I(2010-11) Wolf, Daniel AdamMucopolysaccharidosis type I (MPS I) is caused by deficiency of the lysosomal hydrolase alpha-L-iduronidase (IDUA). IDUA is a required component of the step-wise degradative pathway responsible for the catabolism of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. As a result, these GAGs accumulate within lysosomes causing the development of multisystemic disease. Patients with MPS I present with clinical manifestations of disease within the first two years of life including corneal clouding, hepatoslenomegaly, skeletal dysplasias, cardiopulmonary disease, and obstructive airway disease. Additionally, patients with severe MPS I, also known as Hurler syndrome, develop hydrocephalus and severe neurocognitive decline. The current standard of care for Hurler patients included intravenous administration of recombinant enzyme upon diagnosis followed by hematopoeitic stem cell transplantation (HSCT) once a proper donor cell source is identified. Following HSCT, many patients exhibit a reduced rate of neurological deterioration. However, the potential of HSCT to ameliorate central nervous system manifestations of disease is limited by the inability of IDUA to efficiently cross the blood brain barrier. The results of my experiments demonstrate that GAG storage materials were partially reduced in the brains of MPS I mice following bone marrow transplantation with wild-type donor marrow (Chapter 2). However, pathogenic accumulation of GM3 ganglioside, not normally expressed in the brain, remained present in treated animals. This highlights the necessity to achieve more efficient delivery of IDUA to the central nervous system in order to normalize brain biochemistry. Thus, I propose the application of intracerebroventricular (ICV) infusion of adeno-associated viral vectors in order to mediate gene transfer and expression of IDUA in the brain. Infusion of an AAV serotype 8 vector into neonatal MPS I mice resulted in widespread long-term expression of high levels of IDUA throughout the brain consistent with normalization of GAG storage material and complete prevention of a neurocognitive deficit in a Morris water maze test (Chapter 3). Infusion of the same vector into adult MPS I animals resulted in low levels of IDUA expression and partial reduction of storage material in the brain consistent with partial improvement in the Morris water maze test (Chapter 4). The results of these experiments support the adoption of ICV infusion of AAV vectors as a supplement to enzyme replacement therapy and HSCT for the treatment of Hurler syndrome.Item Novel precise genome editing technologies(2020-09) Aird, EricGenome engineering, the ability to manipulate and modify genomes, has become a standard tool in life sciences and beyond. Programmable nucleases such as CRISPR-Cas9 have afforded the ability to target particular regions in genomes to make targeted changes. While genome editing technologies continue to flourish, the ability for CRISPR-Cas9 to deliver precise genomic modifications is in part hindered by the lower efficiencies of homology-directed repair (HDR). Additionally, delivery of these genome modifying reagents is hampered by current technological constraints. This dissertation describes our unique approaches to developing tools to improve both of these aspects of genome engineering. HUH endonucleases are a family of ssDNA binding proteins that bind sequence specifically to its target and have been co-opted for biotechnological purposes. We developed a Cas9-HUH fusion that increases precise editing outcomes on the order of 2- to 3-fold through covalent tethering of the HDR template. We also demonstrate application of this platform in concert with the development of a molecular tension sensor based on bioluminescent energy transfer (BRET). Next, we pivot to improving delivery of genome editors utilizing adeno-associated virus (AAV). We describe efforts to specifically target AAV to explicit cell types of interest via HUH-antibody selectivity. Building upon this method, we present a novel iteration of a next generation CRISPR-Cas9 based technology termed prime editor. We developed a split prime editor capable of being delivered by dual AAVs in vivo. Overall, the technologies and methodologies developed in this dissertation can readily be incorporated in various CRISPR-Cas workflows to enhance precise genome editing or specific targeting of various genome modifying reagents such as prime editor.Item A synthetic approach to produce recombinant adeno-associated virus (rAAV) in Chinese hamster ovary (CHO) cells(2023-09) Cai, WenRecombinant adeno-associated virus (rAAV) is one of the most promising gene delivery vectors for somatic gene therapy. Currently, its prevailing manufacturing technologies are relying on transiently transfecting host cells with three plasmids or infection of producer cell lines with helper viruses. Both methods pose real issues in process development, such as difficulty to scale-up or cleaning up helper virus from final products. Commonly used host cell lines for rAAV manufacturing are HEK293, and Sf9. Our lab has previously designed a helper virus-free, plasmid-free, stable cell line production system for rAAV2 via synthetic biology approach. The stable cell line was constructed by integrating multiple copies of rAAV2 genome, AAV2 Cap, AAV2 Rep and Ad5 helper genes which are under inducible promoter control and organized as three separate segments in Genome module, Replication module and Packaging module, into HEK293 cells genome. The stable cell line produced infectious rAAV2 particles upon induction. In this study, we aimed to explore the possibility of using Chinese hamster ovary (CHO) cell as the host cell and creating a stable producer cell line for rAAV2 production. Compared to HEK293 and Sf9, CHO cell holds many advantages. As the most commonly used industrial cell line for therapeutic protein production, it could reach high density in suspension cell culture using serum-free media and is resilient and robust in manufacturing conditions. This study showed that CHO cells were capable of translating AAV2 viral proteins, replicating rAAV genomes, and packaging them into rAAV vector in transient transfection using the Genome, Replication and Packaging modules. Expression of two Ad5 gene, E1A and E1B, could further enhance rAAV titer. E1A and E1B could be stably integrated into the CHO-K1 host cell genome along with the three modules under inducible promoter control. Current producer cell lines had a low productivity and their productivity appeared unstable. Nevertheless, our study demonstrated the potential of CHO cell lines as a novel production platform for rAAV manufacturing.