Browsing by Subject "Gene therapy"

Now showing 1 - 7 of 7
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    Characterization of the Sleeping Beauty transposon system for gene therapy applications /
    (2008-05) Score, Paul Rodrick
    Science and medicine are merging in development of gene therapy techniques that will likely become a recognized method for the treatment of human disease within our lifetime. The basic goal of gene therapy is to deliver therapeutic genes to target cell populations, allowing the cells themselves to generate a therapeutic gene product, for example to provide a gene product which is missing in the patient. Most of the current gene therapy trials have been based on the use of viral vectors to deliver therapeutic genes. These viral vectors are being used because they have already evolved mechanisms to efficiently enter cells. Because of the complications inherent in the use of viruses for delivery, a nonviral approach would have several advantages if it could achieve similar levels of efficiency. To this end, I set forth to characterize the Sleeping Beauty (SB) transposon system as a potential tool for effective nonviral gene transfer and its eventual use in clinical gene therapy protocols. The SB transposon system consists of a DNA cargo, usually plasmid based, which in the presence of the transposase integrates into chromosomal DNA. One of the key concerns for any gene delivery system is its ability to function in cells that are not dividing, as many cells in the body that are potential targets for gene therapy are non-dividing. In a series of in vitro experiments utilizing various techniques to halt cell division, I determined that cell division is likely not necessary for SB-mediated integration and expression to occur. Secondly, when tracking expression in vivo, it is not possible to distinguish the amount of gene product produced from integrated vs. nonintegrated transposons. Differentiating between these two sources of transgene expression soon after delivery, will allow insight into transposition efficiency in vivo that can relate to its clinical use. Using LoxP recombination sites, a Cre recombinase inducible mouse strain and transposons carrying a murine erythropoietin gene (Epo), I was able to silence expression from nonintegrated transposons and quantify in vivo gene expression specifically from transposed sequences. Over-expression of erythropoietin in the murine model became an unexpected problem due to subsequent erythrocytosis. Delivery of plasmid DNA to the livers of mice results in an initial spike of transgene expression and when coupled with the subsequent ubiquitous expression of the Epo transgene, circulating Epo levels remained greatly elevated, leading to serious health complications and death. To circumvent the initial spike of EPO expression, an inducible promoter was constructed that responds to hypoxic conditions. In this way, expression of erythropoietin should be regulated to prevent over-expression. Insights gained from these studies will contribute to our understanding of the capabilities of the SB system and its potential application to the treatment of human disease in the future.
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    Gene therapy strategies for targeting the treatment refractory sites in Hurler syndrome.
    (2009-03) Osborn, Mark John
    The submitted work details the development of a novel gene therapy vector capable of expressing multiple genes from a single transcript. This vector allows for high level therapeutic gene expression that is coupled to a dual reporter system that allows for real time in vivo tracking of gene expression as well as cellular detection without need for antibody staining. Additionally, a fusion protein was designed to specifically target the α- L -iduronidase protein to the central nervous system by way of the transferrin receptor. This treatment resulted in a decrease in glycosaminoglycan storage material in the brain of mucopolysaccharidosis type I mice. Lastly, we implemented the use of an episomally maintained plasmid-based vector that mediates high levels of protein production in vivo over a long period of time. Cumulatively this work has generated novel findings that will contribute to the field of gene therapy as a whole.
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    Improving Delivery of Therapeutics to Targeted Tissues in Lysosomal Diseases
    (2021-12) Kim, Sarah
    Lysosomal diseases are a group of over 70 diseases with a combined incidence of approximately 1 in 7,700 live births. Most lysosomal diseases are caused by mutations in enzymes normally present in the lysosome. Lysosomal diseases are multi-systemic and progressive diseases. Currently, only 12 lysosomal diseases have treatments. A challenge in drug development is the lack of biomarkers that reflect disease progression or show response to therapy. Furthermore, current therapies have difficulty reaching certain tissues that are major contributors to morbidity and mortality, such as the central nervous system (CNS) and cardiac valves. The overall objective of this dissertation is to improve the delivery of therapeutics to targeted tissues in lysosomal diseases. To accomplish this objective, three main studies were performed. 1) Validation of Chitotriosidase as a CNS Biomarker for Gangliosidoses. Chitotriosidase was investigated as a probable surrogate endpoint for clinical trials with gene therapy. The first objective was to validate chitotriosidase levels for important clinical outcomes in patients with lysosomal diseases. The second objective was to assess chitotriosidase’s ability to detect effective gene therapy in murine models of lysosomal diseases. In patients with gangliosidoses, the most severe infantile phenotype had higher chitotriosidase levels in the cerebrospinal fluid (CSF) and a different pattern over time than the more attenuated juvenile and late-onset forms. Chitotriosidase levels were also significantly associated with neurocognitive impairment. In mice with mucopolysaccharidosis type I (MPS I), there were significant differences among the untreated, gene-therapy treated, and mice heterozygous for a mutation in the IDUA gene. These results support the use of CSF chitotriosidase levels to diagnose different disease phenotypes and to monitor disease progression in patients. As a potential biomarker of neurological improvement, CSF chitotriosidase can aid in the development of therapies that target the CNS. 2) Investigation of Iduronidase Enzymes Linked to Pepcan to Improve Delivery to Targeted Tissues. The objective of this study was to determine if pepcan-12 can increase the uptake of iduronidase into the brain of MPS I mice. Pepcan-12 is a ligand for the cannabinoid receptor type 1 (CB1), a highly expressed receptor in the CNS. The hypothesis was that a fusion iduronidase containing pepcan-12 would have higher activity levels than iduronidase in the brain. Sequences of one of two linkers, Linker S or Linker T, were inserted between pepcan-12 and IDUA to conjugate the ligand and iduronidase. MPS I mice were injected with plasmids encoding either the native iduronidase or one of four fusion iduronidase enzymes containing: pepcan-12 + Linker S, pepcan-12 + Linker T, Linker S, or Linker T. The fusion enzymes and iduronidase had similar activity levels in the brain. Unexpectedly, the fusion enzymes had higher activity levels than iduronidase in the heart and plasma, which appears to be caused by the linkers. Therefore, these fusion enzymes may improve cardiovascular outcomes in MPS I. In several MPS disorders, the cardiac valves continue to worsen despite enzyme replacement therapies (ERT) and hematopoietic cell transplants. The small size of these linkers facilitates their use as fusion enzymes encoded in gene therapy or administered directly as ERT. Therefore, these linkers may aid in therapeutic development for other lysosomal diseases. 3) Pharmacokinetic Analysis of Iduronidase and a Fusion Iduronidase Enzyme Encoded in Gene Therapy. In the previous study, an iduronidase enzyme containing Linker T, termed Linker T iduronidase, had higher activity levels than iduronidase in the plasma and heart. This study sought to investigate the mechanism of Linker T iduronidase, but a gap between the fields of lysosomal diseases/gene therapy and pharmacokinetics (PK)/ pharmacodynamics (PD) became apparent. In the field of lysosomal diseases, the activity level of an enzyme is an important measurement of efficacy, because an enzyme’s activity levels are more predictive of efficacy than its physical levels. However, pre-clinical studies in lysosomal diseases lack well-described methods to quantify changes in enzyme activity levels over time. In contrast, the pharmacokinetic field has rigorous and reproducible methods to quantify changes in a therapy over time in the body. However, traditional pharmacokinetic methods face challenges in gene therapy because of the need for uniform or convertible units. Furthermore, absorption, distribution, metabolism, and elimination processes are well-characterized for small molecule drugs but not yet adapted for biological therapies. To bridge the fields of lysosomal diseases/gene therapy and PK/PD, I aimed to develop an approach incorporating values with greater prediction of efficacy from the field of lysosomal diseases and the quantitative methods from the field of pharmacokinetics. The objective of this study was to perform a pharmacokinetic analysis of iduronidase and Linker T iduronidase administered as gene therapies. The hypothesis was that Linker T iduronidase would have a higher area under the curve (AUC) or half-life, estimated with enzyme activity levels, than iduronidase in the plasma. MPS I mice were injected with plasmids encoding either iduronidase or Linker T iduronidase. At ten time points, ranging from 0.5 to 168 hours post-injection, the enzymes’ physical levels in the liver, activity levels in the liver, and activity levels in the plasma were measured. In the liver, both the physical and activity levels over time were similar between the native iduronidase and Linker T iduronidase. In contrast, enzyme activity levels over time in the plasma showed differences between the native iduronidase and Linker T iduronidase. The time curves of activity in the plasma showed biphasic profiles for both enzymes. Iduronidase had a sharper decline between 24 and 48 hours, and both enzymes had approximately parallel slopes between 96 and 168 hours. The Linker T iduronidase had a two-fold higher AUC of activity than the normal iduronidase in the plasma. The AUC of plasma activity and other PK parameters were contextualized in gene therapy, and experimental data were used to deduce the mechanism of Linker T. These results suggest that Linker T iduronidase may have a distinct property that protects the enzyme from degradation or inactivation in the plasma. The enzymes were estimated to have a half-life of activity in the plasma under noncompartmental analysis. Future studies with compartmental analysis would better characterize half-lives of activity in biphasic profiles. This study performs a novel approach of conducting a formal pharmacokinetics analysis on enzyme activity levels, a traditionally pharmacodynamic outcome. The resulting PK parameters can be interpreted and used to gain mechanistic insight on gene therapy, by integrating concepts from pharmacokinetics and gene therapy In summary, these findings improve the therapeutic delivery in lysosomal disease through the validation of a CNS biomarker for lysosomal diseases, creation of a fusion enzyme with improved activity in the heart and plasma, and a novel approach and interpretation of pharmacokinetics to gain mechanistic insight on gene therapy.
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    Investigating The Interactions Of Polycations With Nucleic Acid And The Mechanisms Of Delivery
    (2015-05) Sprouse, Dustin
    Polymers - large macromolecules composed from many smaller subunits - have ever-growing uses and potentials in our lives. More specifically, cationically charged polymers have been widely explored as non-viral vectors to deliver nucleic acid to cells in an effort to regulate gene and protein expression. The polymeric vehicle must not only bind and complex with DNA to protect and deliver it to the targeted site, but also efficiently dissociate from the DNA and be non-toxic to the cell or host organism. Herein lies the wide array of polymers that must be rationally designed and synthesized in order for the delivery vehicle to perform its specific function. At the turn of the century, two monumental achievements paved way for gene therapy. First, the Human Genome Project was completed. This milestone continues to unravel important information about the genetic basis of human health, disease, hereditary, and genetic dispositions. Second, RNA interference was discovered, an innate cellular pathway to control gene expression within cells. These discoveries afforded scientists the information necessary to move forward with controlling gene and protein expression profiles. More recently, CRISPR-cas technology was discovered, which allows scientists to permanently edit the genetic code by either regulating genes or adding, disrupting, deleting, or altering the specific base-pairs within the DNA sequence. Herein, we investigate several classes of polymers and macromolecules for the complexation and delivery of nucleic acid, including: amino acids, dendrimers, micelles, and linear homo- and block polymers. Initially, it was shown that polymer type, length, charge, dispersity, and composition greatly affect the efficacy of these therapeutic delivery vehicles. With this in consideration we set out to explore some of the fundamental properties of polymeric vectors. Diblocks, triblocks, and statistical copolymers were designed and synthesized with varying amounts of primary and tertiary amines. These were complexed with pDNA to from polyplexes and probed for their toxicity, stability, gene expression profiles, and mechanisms of membrane permeability. Amphiphilic polymers were also synthesized, which in aqueous environments spontaneously self assembled into core-shell structured micelles. These were probed for their ability to change size in different buffers and form different sized aggregates with DNA.
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    Molecular therapy for mucopolysaccharidosis Type I
    (2010-11) Wolf, Daniel Adam
    Mucopolysaccharidosis 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.
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    Rational design of loss-of-function phospholamban mutants to tune SERCA function.
    (2012-04) Ha, Kim N.
    Unphosphorylated phospholamban (PLN) is the endogenous inhibitor of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA), the enzyme that regulates cardiac muscle relaxation in humans. In its phosphorylated state, PLN (pS16-PLN, pT17-PLN, and pS16pT17-PLN) does not inhibit SERCA. Dysfunctions in SERCA:PLN interactions and in the PLN phosphorylation mechanism have been implicated in cardiac disease and targeting PLN is becoming a viable avenue for treating heart disease. Specifically, innovative genetic treatments using recombinant adeno-associated virus (rAAV) with S16E-PLN, a pseudo-phosphorylated form of PLN, have shown a remarkable efficacy in reducing the progression of cardiac failure in both small and large animals. The following thesis summarizes efforts to rationally design PLN mutants to tune SERCA function. Using a combination of NMR spectroscopy and biochemical assays, we have built a structure-dynamics-function correlation that shows PLN can be tuned to augment SERCA function by acting on the conformational coupling between the cytoplasmic and transmembrane domain and by pseudo-phosphorylation. Additionally, to better understand the role of mutation in PLN:SERCA interactions, we also investigated a mutant of PLN (R9C) known to be linked to hereditary dilated cardiomyopathy, showing that the mutation disrupts the pentamer-monomer equilibrium, and that these effects are exacerbated under oxidizing conditions. Insights to these issues will provide better paradigms with which to design therapeutic mutants of PLN for treatment of heart failure.
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    Trafficking and efficacy of cationic polymers as DNA vaccine carriers and anti-cancer agents
    (2013-06) Panus, David
    The potential of DNA vaccines for treatment of diseases such as HIV and cancer are overwhelming, due to the fact that DNA vaccines can activate both a cell-mediated (T-cell) and humoral (antibody) immune response. However, the most commonly occurring problem of DNA vaccines is limited transgene delivery and expression. Currently, much effort has focused on designing an optimal polymer system that is stable, can protect and deliver DNA, as well as offer high transgene expression. Unfortunately, the ability of polymer based systems to produce robust gene expression, have yet to show substantial improvement. The major obstacle hindering successful transgene expression can be attributed to the interactions of the polymer-DNA complex with the subcellular environment. Therefore, we focused on understanding the structure-functional relationship of a well-defined simple polymer based system and how they might lead to improved transgene expression. First, we investigated the effect of polymer molecular weight and backbone structure on transgene expression as it pertains to subcellular trafficking. Second, we focused on the relationship between polymer-DNA complexes and different dendritic cell-types as a function of maturation state. Lastly, we looked into how further modification of a cationic polymer can lead to elevated cytotoxicity and use as an anti-cancer agent. The results from this work can be used as a design template to improve the overall subcellular trafficking and efficacy of cationic polymer based DNA vaccines or anti-cancer agents.