Browsing by Subject "Gene therapy"

Successful transfection of genetically active materials is essential to gene delivery and genome editing. Electrotransfection, also known as electroporation, is a fast, safe, and convenient non-viral method for introducing materials such as proteins and nucleic acids into cells and tissues. It has been widely used in academic research, industrial manufacturing, and clinical therapeutics. Particularly, electrotransfection is one of the most commonly used method in gene delivery into mammalian cells. However, despite its many advantages comparing to other gene delivery methods, the application of electrotransfection is limited by inconsistent transfection efficiency, which is caused by the poor understanding of the mechanism of electrotransfection.

The goal of my research is to understand the fundamental biological mechanisms of electrotransfection and to develop novel strategies that can improve the transfection efficiency of gene delivery and genome editing. To this end, this study is divided into two phases. Phase 1 aims at understanding the key cellular components involved in the transport process. Phase 2 focuses on the development of strategies to enhance electrotransfection by controlling the biological pathways that are involved in electrotransfection.

In the first phase of my study, we investigated the dependence of electrotransfection efficiency on endocytosis. Data from this study demonstrated that macropinocytosis is involved in electrotransfection. The results revealed that electric pulses induced cell membrane ruffling and actin cytoskeleton remodeling. Using fluorescently labeled pDNA and a macropinocytosis marker (i.e., dextran), the study showed that electrotransfected pDNA co-localized with dextran in intracellular vesicles formed from macropinocytosis. Furthermore, electrotransfection efficiency was reduced significantly by lowering temperature or treatment of cells with a pharmacological inhibitor of Rac1 and could be altered by changing Rac1 activity. Taken together, the findings suggested that electrotransfection of pDNA involved Rac1-dependent macropinocytosis.

Second phase of this study focuses on the intracellular transport of plasmid DNA, especially the transport of DNA molecules towards degradative compartments. Our data elucidated that components in both endocytic and autophagic pathways are responsible for intracellular trafficking and processing of transfected materials such as pDNA. In addition, we also characterized a new type of vesicle named amphisome-like vesicle (ALB) and revealed its involvement in electrotransfection. Based on these findings, we propose a novel strategy to enhance electrotransfection by blocking degradative routes within the endocytic pathways, which led to the development of a new technique called transfection by redirection of endocytic and autophagic traffic (TREAT). Transfection of plasmid DNA (pDNA), messenger RNA (mRNA), sleeping beauty transposon system (SB), and different forms of CRISPR/Cas9 system by TREAT achieved superior efficiency in various cell lines including difficult-to-transfect human primary cells. In addition, we successfully applied TREAT method to improve clinically relevant applications including SB-based gene integration and CRISPR/Cas9-based editing of T cell receptor alpha constant (TRAC). In summary, we studied the biological mechanism of electrotransfection and developed a general, flexible, and reliable technique to enable highly efficient non-viral gene delivery and genome editing. Furthermore, the insights gained on the mechanism of electrotransfection provide better understanding of cellular response to exogenous materials. In the future, our study could potentially pave new paths for a wide range of research and therapeutic applications such as CRISPR/Cas9 mediated high-throughput loss-of-function gene screening analysis, correction of disease-related mutations, as well as genetic engineering of immune cells and stem cells for transplantation.

Therapeutic gene transfer and genome editing require effective delivery of genetic cargo to target cells and tissues. Recombinant adeno-associated viral (AAV) vectors are a promising delivery platform, but ongoing clinical trials continue to highlight a relatively narrow therapeutic window. Efforts to optimize vector dosing or engineer improved vectors are confounded, at least in part, by differences in AAV biology across animal species. Here, we present a broadly applicable, cross-species evolution approach to tackle this challenge. Specifically, I iteratively cycled AAV libraries administered intravenously and amplified isolates from CNS tissue in pigs, mice, and non-human primates to generate cross-species compatible AAVs (ccAAVs). By sequentially evolving AAV libraries in three different species, we discover a highly potent variant (AAV.cc47) that demonstrates improved attributes benchmarked against AAV serotype 9 (AAV9). Increased potency of AAV.cc47 is evidenced through robust reporter gene expression as well as Cre-mediated recombination and CRISPR/Cas9-mediated genome editing in a fluorescent reporter mouse model. 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. Lastly, we report increased expression of a therapeutic acid alpha-glucosidase (GAA) transgene in a mouse model of Pompe disease and enhanced restoration of dystrophin through CRISPR/Cas9 gene editing in the mdx mouse model of Duchenne Muscular Dystrophy using AAV.cc47 vectors. We discovered another cross-species compatible variant (AAV.cc84) with enhanced CNS transduction and de-targeted from the liver in vivo compared to AAV9. We demonstrate improved targeting of neurons in the brain following both systemic and intracerebroventricular (ICV) injection of reporter vectors in mice. Reporter gene expression and vector genome biodistribution reveal a liver detargeted phenotype with AAV.cc84 compared to AAV9. Lastly, enhanced neuronal transduction was confirmed in the pig CNS following intrathecal infusion of AAV.cc84 reporter vectors. Taken together, we envision that ccAAV vectors can potentially improve predictive modeling in preclinical studies as well as clinical translatability by broadening the therapeutic window of AAV based gene therapies.

Genetic modification of dendritic cells with plasmid DNA is plagued with low transfection efficiencies because DNA taken up by non-dividing dendritic cells rarely reaches the nucleus. But this difficulty can be overcome by the use of messenger RNA (mRNA), which exerts its biological function in the cytoplasm and obviates the need to enter the nucleus. Since pioneering work of Boczkwoski et al, the ex-vivo application of mRNA-transfected dendritic cells as a vaccine has been evaluated in numerous phase I trials worldwide and is still currently being actively optimized in clinical trials.

However, a major disadvantage of using mRNA-transfected DCs as a vaccine is that it requires patients to undergo at least one 4-hour leukapheresis procedure, followed by separation of the peripheral blood mononuclear cells (PBMCs), from which monocytes are isolated and cultured for a week in a defined medium with cytokines. The resulting DCs are matured after being loaded with mRNA and frozen for storage. Aliquots are subsequently thawed prior to administration to patients. This process of harvesting, culturing and loading DCs is more time- and resource-intensive than Provenge, the first FDA approved cell based tumor vaccine in 2011.Recent evidence has confirmed a lack of broad translation of Provenge due to complexity and cost of treatment. This predicates a similar fate for mRNA-transfected dendritic cell vaccine going forward.

This thesis presents alternative delivery strategies for mRNA mediated tumor vaccination. Through the application of synthetic and natural biomaterials, this thesis demonstrates two viable approaches that reduce or eliminate the need for extensive manipulation and cell culture.

The first approach is the direct in vivo delivery of mRNA encapsulated in nanoparticles for tumor vaccination. A selected number of synthetic gene carriers that have been shown to be effective for other applications are formulated with mRNA into nanoparticles and evaluated for their ability to transfect primary DCs. The best performing formulation is observed to transfect primary murine and human dendritic cells with an efficiency of 60% and 50% (based on %GFP+ cells) respectively. The in vivo transfection efficiency and expression kinetics of this formulation is subsequently evaluated and compared with naked mRNA via various routes of delivery. Following this, a proof-of-concept study is presented for a non-invasive method of mRNA tumor vaccination using intranasally administered mRNA encapsulated in nanoparticles. Results show that intranasally administered mRNA induces tumor immunity only if it is encapsulated in nanoparticles. And anti-tumor immunity is observed in mice intranasally immunized under both prophylactic as well as therapeutic models.

The second approach evaluates whole blood cells as alternative cell based mRNA carriers. A method is developed to encapsulate intact and functional mRNA in murine whole blood cells. Whole blood cells loaded with mRNA not only include erythrocytes but also T cells (CD3+), monocytes (CD11b), antigen presenting cells (MHC class II) as well as plasmacytoid DCs (CD45R-B220). Mice immunized with mRNA-loaded whole blood cells (intravenously) develop both humoral and cellular antigen-specific immune responses, and demonstrate delayed tumor onset and progression in a melanoma therapeutic immunization model (using tyrosinase related protein -2, TRP-2, as an antigen). Importantly, the therapeutic efficacy of mRNA-loaded whole blood cell vaccine formulation is found to be comparable to mRNA-transfected dendritic cell vaccine.

In conclusion, this thesis presents new methods to the delivery of mRNA tumor vaccines that reduce or eliminates the need for extensive cell manipulation and culture. Results presented in this thesis reveal viable research directions towards the development and optimization of mRNA delivery technologies that will address the problem of broad translation of mRNA tumor vaccines in the clinics.

Glycogen storage disease type Ia (GSD Ia) is an autosomal recessive metabolicdisorder caused deficiency of glucose-6-phosphatase (G6Pase) resulting from pathogenic variants in the G6PC gene. G6Pase catalyzes the hydrolysis of glucose-6-phosphate to release glucose which can then enter the bloodstream. GSD Ia patients have excess glycogen accumulation mainly in the liver and kidneys and suffer from life threatening hypoglycemia. The current treatment for GSD Ia is dietary therapy that requires patients to frequently consume uncooked cornstarch on a strict schedule. Cornstarch provides a complex carbohydrate that slowly releases glucose to prevent hypoglycemia. This treatment fails to prevent long-term complications associated with GSD Ia including renal failure and the development of hepatocellular adenomas and carcinomas. This lab and others have developed adeno-associated virus (AAV) vector based gene therapies to deliver and therapeutic G6PC transgene to affected tissues in GSD Ia animal models. However, the therapeutic effect is limited as AAV vector genomes are rapidly lost and the biochemical correction declines. Currently no treatment for GSD Ia exists that provides stable, robust expression of G6Pase that can clear glycogen and prevent hypoglycemia. This study employed a novel genome editing approach designed to insert the therapeutic G6PC into the endogenous locus in canine and murine models of GSD Ia. Integration of the transgene into the genome will promote stable expression of G6Pase and prevent the decline of vector genomes and the therapeutic benefit. This genome editing approach utilizes the CRISPR/Cas9 system to generated targeted double stranded DNA breaks at a targeted site in the genome. The G6PC transgene is present in a Donor template with homology to the DNA break to drive homology directed repair (HDR) resulting in the integration of the transgene into the genome. In a canine model of GSD Ia, editing and incorporation of the transgene was achieved in both adult dogs and puppies. Up to 1.0% of alleles were edited in the dog livers and contained the transgene. G6Pase production from the integrated transgene was detected, which correlated with prevention of hypoglycemia during fasting. This demonstrated genome editing in the liver of a large animal model for an inherited metabolic disorder using HDR to insert a therapeutic transgene. A subsequent study in GSD Ia mice also showed incorporation of a G6PC transgene in the mouse genome. Mice were treated with either the Donor transgene vector alone or with both the Donor and a CRISPR/Cas9 vector to assess to role of nuclease activity on integration. Mice treated with both vectors demonstrated improved blood glucose concentrations during fasting, decreased liver glycogen, and increased vector genome copies. Treatment with the pan PPAR agonist bezafibrate increased the efficiency of genome editing. Mice treated with bezafibrate that received both editing vectors had 5.9% of alleles that contained the integrated transgene, whereas only 3.1% of alleles contained the transgene in mice not treated with the drug. This work showed that integration of a therapeutic transgene using CRISPR/Cas9 based genome editing is possible in murine and canine models of GSD Ia. Editing resulted in biochemical correction and sustained transgene expression. These data support the further development of genome editing technologies for GSD Ia and other inherited metabolic disorders.

Recombinant adeno-associated virus (AAV) has emerged as a leading platform for therapeutic gene transfer. Despite significant progress and a string of FDA approvals, significant challenges that prevent the broader application of AAV gene therapy exist. High systemic dosing of AAV vectors in the clinic poses potential risk of severe and adverse side-effects due to anti-capsid immunity. While different immune modulatory regimens (IMR) are being explored, there is an urgent need for continued development of effective strategies to improve the safety of AAV gene therapies. In chapter 2 of this dissertation, I describe the discovery of a novel enzyme (IceM) that cleaves human IgM, a key trigger in the anti-AAV immune cascade. We then engineer a fusion enzyme (IceMG) with dual proteolytic activity against human IgM and IgG. IceMG cleaves B cell surface antigen receptors (BCR) and inactivates phospholipase gamma signaling in vitro. Importantly, IceMG is more effective at inhibiting complement activation compared to an IgG cleaving enzyme alone. Administration of IceMG in rhesus macaques enables robust and reversible clearance of both circulating IgM and IgG. Antisera from macaques treated with IceMG shows significantly decreased AAV neutralization as well as complement activation. Finally, we demonstrate that pre-treatment with IceMG restores AAV vector transduction in mice passively immunized with human antisera containing anti-AAV neutralizing antibodies. Thus, IgM cleaving enzymes show promise in simultaneously addressing multiple aspects of anti-AAV immunity mediated by B cells, circulating antibodies and complement. These studies have implications for improving safety of AAV gene therapies and more broadly, for use in organ transplantation and autoimmune diseases. In parallel, unraveling the biology of AAV entry and trafficking is central to developing improved AAV vectors. In chapter 3 of this dissertation, I delve into the biology of host factors that limit AAV transduction. We identify furin as a host factor that significantly restricts the transduction of AAV4-like serotypes. Through the interrogation of different steps and attachment factors in the AAV infectious pathway, we demonstrate that AAV cellular binding and uptake are significantly increased in a sialic acid-dependent manner. We postulate that furin likely plays a key role in regulating expression of cellular sialoglycans, which in turn, can influence permissivity to AAVs and possibly other viruses.

Synthetic small diameter vascular grafts fail clinically due to thrombosis and intimal hyperplasia. The attachment of endothelial cells (ECs) onto the inner lumen of synthetic small diameter vascular grafts can improve graft patency; however, significant challenges remain that prevent wide clinical adoption. These issues include difficulties in the autologous sourcing of ECs, the lack of attachment, growth and retention of the layer of ECs to the graft lumen, and the maintenance of an anti-thrombotic and anti-inflammatory profile by the layer of ECs.

This dissertation describes the isolation, characterization, and use of endothelial progenitor cells (EPCs) to improve the performance of small diameter vascular grafts. First, EPC isolation efficiency and expression of critical EC markers was compared between young healthy volunteers and patients with documented coronary artery disease (CAD). EPCs were isolated and expanded from patients with CAD and had a similar phenotype to EPCs isolated from healthy donors, and a control population of human aortic ECs. Second, we assessed the ability to enhance the anti-thrombotic activity of patient derived EPCs through the over expression of thrombomodulin (TM). In vitro testing showed TM-transfected EPCs had significantly increased production of key anti-thrombotic molecules, reduced platelet adhesion, and extended clotting times over untransfected EPCs. Finally, native and TM-transfected EPCs were seeded onto small diameter vascular grafts and tested for their ability to improve graft performance. EPCs sodded onto the lumen of small diameter ePTFE vascular grafts had strong adhesion and remained adherent during graft clamping and exposure to flow. TM-transfected EPCs improved graft anti-thrombotic performance significantly over bare grafts and grafts seeded with native EPCs. Based on these promising in vitro results, grafts were implanted bilaterally into the femoral arteries of athymic rats. Bare grafts and grafts with air removed clotted and had only 25% patency at 7 days. In contrast, graft sodded with native EPCs or TM-transfected EPCs had 87% and 89% respective patency rates. High patency rates continued with 28 day implant testing with EPC sodded grafts (88% Native; 75% TM). There were no significant differences in patency rates at 7 or 28 days between native and TM-transfected grafts. These in vivo data suggest patient blood-derived EPCs can be used to improve the performance of small diameter vascular grafts.

Post-traumatic arthritis (PTA) is arthritis that develops following joint injury, including meniscus and ligament tears. Current treatments for PTA range from over-the-counter medication to knee replacement; however, in the presence of obesity, the levels of pro-inflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α,) are more elevated than in non-obese individuals. The role of fatty acids, obesity, and PTA has been examined, with omega-3 fatty acids showing promise as an anti-inflammatory after injury due to its ability to suppress IL-1 and TNF-α. Due to the difficulty in switching patients’ diets, an alternative solution to increasing omega-3 levels needs to be developed. The Fat-1 enzyme, an omega-3 desaturase that has the ability to convert omega-6 to omega-3 fatty acids, may be a good target for increasing the omega-3 levels in the body.

In the first study, we examined whether Fat-1 transgenic mice on a high-fat diet would exhibit lower levels of PTA degeneration following the destabilization of the medial meniscus (DMM). Both male and female Fat-1 and wild-type (WT) littermates were put on either a control diet (10% fat) or an omega-6 rich high-fat diet (60% fat) and underwent DMM surgery. Arthritic changes were examined 12 weeks post-surgery. Fat-1 mice on both the control and high-fat diet showed protection from PTA-related degeneration, while WT mice showed severe arthritic changes. These findings suggest that the omega-6/omega-3 ratio plays an important role in reducing PTA following injury, and demonstrates the potential therapeutic benefit of the Fat-1 enzyme in preventing PTA in both normal and obese patients following acute injury.

Following this, we needed to establish a translatable delivery mechanism for getting the Fat-1 enzyme, which is not present in mammalian cells, into patients. In the second study, we examined whether anti-inflammatory gene delivery of the Fat-1 enzyme would prevent PTA following DMM surgery. In vitro testing of both lentivirus (LV) and adeno-associated virus (AAV) was completed to confirm functionality and conformation of the Fat-1 enzyme after transduction. Male WT mice were placed on an omega-6 rich high-fat diet (60% fat) and underwent DMM surgery; either local or systemic AAV injections of the Fat-1 enzyme or Luciferase, a vector control, were given immediately following surgery. 12 weeks post-surgery, arthritic changes were assessed. The systemic administration of the Fat-1 enzyme showed protection from synovial inflammation and osteophyte formation, while administration of Luciferase did not confer protection. These findings suggest the utility of gene therapy to deliver the Fat-1 enzyme, which has potential as a therapeutic for injured obese patients for the prevention of PTA.