Thesis advisor: Thomas N. Seyfried / Gangliosides are glycosphingolipids (GSLs) containing sialic acids that play numerous roles in neuronal maturation, apoptotic signaling, angiogenesis, and cell surface receptor activity. The GM1 and GM2 gangliosidoses are a series of autosomal recessive lysosomal storage disorders (LSDs) characterized by an inability to degrade these lipid molecules. GM1 gangliosidosis is caused by a mutation in the lysosomal hydrolase β-galactosidase, resulting in neuronal storage of ganglioside GM1 and asialo GA1. Tay-Sachs (TS) and Sandhoff Disease (SD) are GM2 gangliosidoses caused by mutations in either the α or β subunits, respectively, of the heterodimeric protein β- hexosaminidase A, resulting in the storage of ganglioside GM2 and asialo GA2. The accumulation of excess ganglioside in the central nervous system leads to abnormal intracellular vacuoles, neuronal loss, demyelination, ataxia, dementia, and premature death. In my studies, I have shown that accumulation of GM1 ganglioside may not coincide with secondary storage of cholesterol, by providing evidence that cholesterol-binding fluorescent molecule filipin reacted to GM1 ganglioside in the absence of cholesterol. In an effort to combat the early-onset gangliosidoses, I have explored the effects of combining Neural Stem Cells (NSCs) with Substrate Reduction Therapy (SRT) in juvenile Sandhoff mice. The analysis showed that SRT was more effective than NSCs in reducing stored GM2 and GA2 in young mice, and no synergy was observed. In adult GM1 gangliosidosis, Tay- Sachs, and Sandhoff mice, Adeno-Associated Viral (AAV) vector gene therapy was used to restore therapeutic levels of wild-type enzyme to the CNS. AAV therapy corrected ganglioside storage and ameliorated myelin-associated lipid loss in all tissues assayed, increasing motor performance and life in effected animals. Lastly, AAV therapy was also successful in a feline model of Sandhoff disease. These results in juvenile and adult model systems point the way towards multiple effective clinical therapies in the near future. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

There is currently no protective vaccine against HIV. It is known that a high mutation rate and the existence of many subspecies or clades generated by point mutations or recombination events, are at least partly responsible for the ability of the virus to escape immune responses elicited by classical vaccines. Protein subunit vaccines may not be effective due to this pronounced viral mutability. An immune evasion mechanism has been postulated in which variable domains occlude conserved epitopes crucial for infectivity. The use of DNA vaccines appeared as a favorable approach. Here, a DNA vaccine approach is presented in which the DNA constructs have been engineered to circumvent the aforementioned problems by 1) introducing elements to enhance expression, such as a heterologous promoter, a heterologous signal sequence and intron sequences, 2) by optimizing codon usage, and 3) by vaccinating with antigens that have a modified glycosylation pattern which will make them more immunogenic. The results indicated that deglycosylation of different clades of gp120 did not affect the protein conformation, and 'in vitro' expression levels were good. Antigen codon optimization dramatically increased antibody production. In the animals vaccinated with non-codon-optimized constructs, the presence of an intron and a heterologous signal sequence was required to achieve a good antibody response. Therefore, antigen engineering is required to obtain a powerful immune response against HIV-1 gp120.

Les vecteurs lentiviraux (LV) sont des outils efficaces de transfert de gène, largement utilisés en thérapie génique, en particulier pour la transduction ex vivo de cellules souches et progénitrices hématopoïétiques (CSPH). Afin d’étudier simultanément la fusion et la transduction dans les CSPH avec les LV, nous avons adapté une méthode basée sur latechnologie du transfert d’énergie entre deux molécules fluorescentes (FRET). Pour mettre en place cette technique, des LV capables d’incorporer spécifiquement une enzyme, la bétalactamase (BLAM-LV) et de coder une forme tronquée du récepteur au facteur de croissance nerveuse (DELTA-NGFR), sont produits. Nos résultats montrent que les LV sont soumis à une restriction post-entrée forte dans les cellules hématopoïétiques, que ce soit dans des lymphocytes T immortalisés ou bien des CSH CD34+. Nous montrons également que cette inhibition post-entrée peut être partiellement saturée après une forte augmentation de la multiplicité d’infection ou en présence d’additifs de culture, comme la Vectofusin-1® ou laRetronectin®. De plus, nous avons montré lors de la transduction de CSPH avec des vecteurs BLAM-LV que la Vectofusin-1® agit sur l’étape d’entrée en augmentant l’adhésion et la fusion entre les membranes virale et cellulaire. Cette technique représente donc un nouvel outil sensible et efficace pour étudier de façon concomitante l’étape de fusion et le niveau de transduction dans les cellules cibles. A terme, ce travail permettra une meilleure compréhension de la biologie des LV mais pourra également conduire à l’élaboration de protocoles de transduction lentivirale plus efficaces. / Lentiviral vectors (LV) are used for various gene transfer applications, notably for hematopoietic gene therapy, but methods are lacking to precisely evaluate parameters that control the efficiency of transduction in relation with the entry of vectors into target cells. We adapted a fluorescence resonance energy transfer (FRET)-based HIV-1 fusion assay to measure the entry of non-replicative recombinant LV in various cell types, including primary human hematopoietic stem and progenitor cells, and to quantify the level of transduction of he same initially-infected cells. The assay utilizes recombinant LV containing betalactamase (BLAM)-Vpr chimeric proteins (BLAM-LV) and encoding a truncated form of thelow affinity nerve growth factor receptor (DELTA-NGFR). This LV-based fusion/transduction assay is a dynamic and versatile tool, revealing for instance the extent of lentiviral post-entry restrictions occuring in cells of hematopoietic origin. The assay also shows that transduction enhancers like Vectofusin®-1 or Retronectin® can partially relieve this post-entry block but their effects differ in the way to promote LV entry. Furthermore, our results show that Vectofusin®-1 acts at the entry step by promoting the adhesion and the fusion between lentiviral and cellular membranes. In conclusion, one such assay should be useful to study hematopoietic post-entry restrictions directed against LV and should allow improvements in various LV-based gene therapy protocols.

No description available.

Due to recent advances in molecular biology and genomic research, numerous diseases have been given their genetic identities for which gene therapy may be a possible prescription. Gradually, the development of viral and non-viral vectors to translocate genes has become a bottleneck. For non-viral vectors, polyethylenimine (PEI) is considered as a potential vector candidate for gene delivery because of its ability to compact DNA and its intrinsic pH buffering capacity. PEI and its derivates have been widely tested in both in-vitro and in-vivo gene transfection experiments. The progress is limited due to the lack of a better understanding of the intracellular mechanism. So far, their cytotoxicity is relatively high and gene transfection efficiency is low. This study was designed to modify PEI and optimize its cytotoxicity and gene transfection efficiency. / During the complexes formation, both LLS and zeta-potential were used to follow the process. The results showed that most of anionic DNA are complexed by cationic PEI-based polymers when the molar ratio of nitrogen from PEI to phosphate from DNA (N:P) reaches ∼3, but the gene transfection reaches the highest efficiency when N:P ∼10. When N:P > 3, there exist two population of PEI chains in the solution mixture: bound to DNA and free in the solution. The bound PEI chains condense and protect DNA. Our current study confirms that it is those free PEI chains that play a vital role in promoting the gene transfection. Our preliminary data shows that the promotion mainly occurs in the intracellular space. The detailed mechanism is still lacking at this moment. Nevertheless, our finding leads to a totally different way in the development of non-viral vectors. / Further, we grafted PEI with polyethylene glycol (PEG), respectively via a reductive disulfide -S-S- and a non-degradable -C-C- bond to form two copolymer vectors. A comparative study shows that the polyplexes formed between the two copolymers and DNA are more stable than that formed between unmodified PEI and DNA under the physiological condition, presumably because the grated PEG chains form a protective hydrophilic shell on the PEI/DNA polyplexes. However, PEGylation reduces the internalization of the copolymer/DNA polyplexes in in-vitro experiments. For the two copolymer vectors, PEG-SS-PEI is 2-8 times more effective than its counterpart (PEG-CC-PEI) in the gene transfection, presumably due to the cleavage of the grafted PEG chains inside the reductive cytosol, which promotes the release and translocation of DNA. Our results demonstrate that using the disulfide as a linker is a promising approach to overcome the PEGylation dilemma in the development of low cytotoxic and high efficient non-viral polymeric vectors. / It has been known that short PEI chains are less toxic, but long chains are more effective in gene transfection. Therefore, we decide to use the disulfide bond (-S-S-) to extend short PEI chains to increase efficiency and also utilize the reductive cytosol environment to cleave such extended PEI chains to reduce their cytotoxicity inside the cell. Laser light scattering (LLS) was used to in-situ monitor the linking reaction between short PEI chains (M w = 2000 g/mol) and dithiobis(succinimidyl propionate) (DSP). The molar mass and crosslinking degree of the extended PEI chains was controlled by either the amounts or the adding rate of DSP. A comparative study of two linked PEI samples (PEI-7K-L and PEI-400K-L, respectively with M w = 6.5 x 103 and 3.8 x 10 5 g/mol) reveals that cytotoxicity and gene transfection efficiency of such extended PEI chains are related to the chain length and structure. Namely, PEI-7K-L with an extended chain structure is less cytotoxic and 2--10 times more effective in the gene transfection than the "golden standard" (PEI25K) and the widely used commercial vector, Lipofectamine 2000RTM. Comparatively, PEI-400K-L with a spherical microgel structure is ineffective in spite of its non-toxicity. Our study clearly demonstrates that a proper control of the chain length and structure is important. / by Deng, Rui. / Adviser: Chi Wu. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Parkinson’s disease remains a major neurodegenerative disease with prevalence that is second only to Alzheimer’s disease. Despite much advancement in the understanding of the pathogenesis of Parkinson’s disease, current therapeutic options are limited to those that are only symptomatic and are not disease-modifying. Furthermore, due to what seems to be increasingly complex underlying mechanisms of the disease, identifying a broadly applicable therapeutic strategy is difficult. There is much evidence surrounding the role of apoptosis and conversely, dysfunction of anti-apoptotic signaling in the progressive neurodegeneration that causes Parkinson’s disease. In particular, suppressed PI3K signaling has been implicated in the literature as a key event that occurs during neurodegeneration. Thus, regardless of the diverse upstream mechanisms that may lead to the disease, targeting a downstream effector of neuronal survival presents a therapeutic strategy that may be broadly effective against Parkinson’s disease. For this dissertation, we have developed a method to control the levels of active Akt, the main mediator of the PI3K signaling pathway, using an innovative protein destabilizing technique to create an inducible Akt, DD-Akt(E40K). This method permits the control of active Akt levels through a commonly used and blood-brain barrier permeable antibiotic, Trimethoprim. We have successfully established the inducibility of DD-Akt(E40K) across various cellular contexts, including neuronal cell types and conditions with suppressed PI3K signaling. This inducibility was found to be dose-responsive to Trimethoprim, reversible, and able to induce a known downstream substrate, FoxO4, that is an important regulator of cell survival. Importantly, DD-Akt(E40K) was found to inducibly protect neuronal PC12 cells against Parkinson’s disease mimetic toxins as well as growth-factor removal, indicating a proof of principle for the targeting of Akt activity as a protective strategy against Parkinson’s disease. The reported trophic effects of active Akt were also corroborated using our inducible DD-Akt(E40K) system in vivo, demonstrating significant increases in neuronal cell size within the substantia nigra of mice. Intriguingly, the inducibility of DD-Akt(E40K) was found to be dependent on the region of expression in the brain of mice such that the levels of this protective protein were not controllable by Trimethoprim in the substantia nigra but were controllable in the striatum. Taken together, the studies presented in this dissertation establish a new tool for the study of Akt signaling in various cellular and disease contexts and validate Akt as a promising therapeutic target in Parkinson’s disease. Our results also suggest an intriguing mechanism for the underlying pathology and selective degeneration observed in the disease.

Spinal muscular atrophy (SMA) is a severe genetic neuromuscular disorder characterised by lower motor neuron degeneration and paralysis. Although it is a leading genetic cause of childhood death no approved treatment options currently exist. As SMA is caused by low levels of the survival motor neuron (SMN) protein the majority of therapeutic strategies under development are therefore aimed at trying to elevate SMN levels. However, a number of limitations with these approaches exist demonstrating a need for the investigation of SMN-independent therapeutics. Of these non-classical pathways, the ubiquitin proteasome system (UPS) is an exciting new area of SMA research. The UPS is a system which degrades unwanted or damaged proteins and alterations in the UPS (including reduced levels of ubiquitin-like modifier activating enzyme 1 [Uba1] and increased levels of ubiquitin carboxyl-terminal esterase L1 [Uchl1] and β-catenin) have been recently identified in the neuromuscular system of SMA mice, providing promising new targets for therapy development. In this thesis I demonstrate that UPS perturbations are also present in other organ systems of severe ‘Taiwanese’ SMA mice and in other SMA models including intermediate Smn2B/− mice, zebrafish and patient derived iPSC motor neurons. Given the previously demonstrated improved neuromuscular phenotype in SMA mice treated with the β-catenin inhibitor quercetin I have been establishing whether other compounds with β-catenin inhibition offer similar or even better therapeutic options. Aspirin, indomethacin and iCRT-14 trials did not improve the SMA phenotype with likely off-target adverse effects meaning that quercetin remains the most tolerable β- catenin inhibitor in SMA mice to date. Another potential target of the UPS for SMA therapeutics is the deubiquitinating enzyme Uchl1, levels of which are increased in SMA. In this thesis I show that pharmacological inhibition of Uchl1 did not improve survival or motor performance in SMA mice and instead had a detrimental impact on the disease phenotype which could be explained by worsening SMA ubiquitin defects. Histological analysis revealed that there was no improvement in lower motor neuron count numbers, neuromuscular junction deficits or muscle fibre diameters. Mimicking the UPS phenotype in primary neuronal cells suggested that targeting UPS perturbations observed in SMA that are upstream of Uchl1, particularly the loss of Uba1, may therefore offer a more effective therapeutic option. Finally, I therefore examined whether increasing Uba1 levels in SMA mice using gene therapy technology was able to improve the SMA phenotype. My initial studies indicate that delivery of AAV9-UBA1 to SMA mice may be beneficial as intraperitoneal injection of AAV9-UBA1 was found to increase the weight and improve motor performance of SMA mice. Intravenous delivery of AAV9-UBA1 was found to further improve expression levels and biodistribution of AAV9-UBA1 in the central nervous system as well as systemically in all body organs and tissues. Western blot and proteomic analysis revealed that AAV9-UBA1 gene therapy is also able to correct downstream UPS perturbations found in SMA as well as increase SMN levels. Together, these results suggest that AAV9-UBA1 gene therapy is an exciting novel therapeutic approach for SMA.

Androgens are essential for the maintenance of male health and wellbeing. A disturbance in androgen signalling has been associated with a number of clinically relevant disorders such as cardiovascular disease, diabetes and metabolic disorders as well as infertility. Primarily produced in the testis in males, the actions of androgens are mediated through binding to androgen receptor (AR), a member of the nuclear receptor superfamily of ligand-activated transcription factors. The somatic cells of the testis are known to have a number of key roles in both testis function and development and the Sertoli, Leydig and Peritubular Myoid cells are known to express AR in adulthood. It is through AR that some testicular functions are mediated; for example, the Sertoli cells support of complete spermatogenesis with Sertoli cell androgen receptor knockout (SCARKO) testis demonstrating a halt of spermatogenesis before meiosis. However, how androgen signalling is impacting testicular function through each of the somatic cell types is not yet fully understood. Currently, treatments for male reproductive disorders such as hypogonadism (low androgens) and infertility are limited to treatment of the symptoms; using androgen replacement therapy and in vitro fertilisation techniques. This has been, up until recently, a result of a lack of understanding of the causes of these conditions and a lack of resources able to treat them, with research suggesting that a genetic component may be responsible in a number of cases. However, due to the limited genetic investigation diagnosis of men with male reproductive disorders, the wider understanding of the genetics underpinning male hypogonadism and infertility is incomplete. Developments in technology for the investigation and editing of the genetic code are triggering a surge in the exploration of genetic disorders and, in parallel, into the fields of gene delivery vectors and editing technologies. These technologies will allow an expansion into the knowledge and understanding of genetic disorders whilst simultaneously affording the opportunity to exploit this understanding for the development of therapeutics. There have been a small handful of previous studies using technologies such as viral vectors to target the testicular somatic cells and deliver exogenous transgenes with the purpose of both gene editing and repair, all with varying degrees of success. Here, techniques to introduce and target the Leydig and Sertoli cells were investigated to determine the most appropriate methodology for gene delivery to and manipulation of the testis. Refinement of injections into the interstitial compartment were carried out before introducing lentiviral vectors and targeting of Leydig cells was validated and optimised. Lentiviral vectors are able to permanently integrate into the host cell. Surprisingly, analysis of testis post lentiviral injection determined that the lentiviral targeted Leydig cells began to undergo apoptosis one week post injection and were subsequently cleared from the testis after ten days. Contrastingly, this was not the case when adenoviral vectors were introduced into the interstitial compartment, with Leydig cells continuing to express the delivered reporter transgene and, importantly, not expressing markers of apoptosis, ten days post injection. This would suggest that using adenoviral vectors to target the Leydig cell population in the adult testis would be more appropriate than using lentiviral vectors. Previous studies have successfully used lentiviral vectors to target the Sertoli cells in the adult testis via the introduction of the particles through the efferent duct. However, this can result in damage to efferent duct, resulting in blockages and subsequently the seminiferous tubules. To circumvent this, introduction of the lentiviral particles through the rete compartment of the testis at a range of lower injection pressures was examined and injecting at a lower pressure through the rete testis was found to reduce the likelihood of introducing negative impacts on testicular histology when targeting the seminiferous tubules. Using these refined methods of introducing lentiviral vectors, targeted Sertoli cells stably expressed the delivered transgene for up to one year post injection. Using viral vector delivered transgenes for both the investigation of testicular genetic disorders and for the development of therapeutics has great potential. To explore this potential, we first generated a mouse model in which AR was ablated from both the Leydig and Sertoli cells using Cre/LoxP technology, termed the SC-LC-ARKO. Alongside providing a potential model to 'repair' with viral vectors, the SC-LC-ARKO model also provided an additional model for comparison with other models exhibiting ablation of AR from both single somatic cell types and double somatic cell types. This further enabled a characterisation of the roles of AR in adult testicular function, with results suggesting that loss of AR from more than one cell type results in an additive phenotype when compared to single cell knock outs. Despite providing further insight into the roles of AR in the testis, further analysis of the Cre line used to generate the SC-LC-ARKO model indicated that a small number of Leydig cells were expressing the Cre recombinase, resulting in only a small population of Leydig cells with ablated AR. Considering this, to explore the potential of rescuing Sertoli cell AR using lentiviral vectors, we then utilised an already well characterised Sertoli Cell AR knockout (SCARKO) model. Lentiviral vectors expressing mouse AR and monomeric GFP (moeGFP) downstream of a CMV promoter were generated and injected into the rete testis of WT and SCARKO adult (day 100) males at low pressure. The contralateral testis was injected with a lentiviral vector expressing moeGFP alone (also downstream of a CMV promoter) using the same technique. Analysis of testis sections revealed a reintroduction of AR to Sertoli cells in 100% of SCARKO testis injected with lentivirus expressing mouse AR. As a result of this re-expression of AR in Sertoli cells, 66% of the testis injected with lentivirus expressing mouse AR had evidence of morphologically mature elongated spermatids, indicative of ongoing spermatogenesis. These results suggest that a rescue of the infertility phenotype reported in previous studies of SCARKO testis. Also demonstrated is the reversal of the SCARKO testicular phenotype in tubules targeted by the mAR expressing lentiviral vector. This suggests that absence Sertoli cell AR throughout development does not have a permanent impact on the Sertoli cells capacity to support spermatogenesis in adulthood following rescue of SC AR expression in adulthood. In summary, the results of these studies have provided a refinement in the methodologies for targeting the Sertoli and Leydig cells of the adult testis with viral vectors as well as demonstrating successful rescue of a previously reported mouse model exhibiting infertility through reintroduction of a functional gene. Alongside this, comparisons of AR knockout models have afforded insight into maintenance of testis function through AR.

Yue, Yanan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 62-63). / Abstract also in Chinese. / ABSTRACT (Chinese) --- p.i / ABSTRACT --- p.iii / CONTENT --- p.v / ACKNOWLEDGMENT --- p.vii / ABBREVIATIONS --- p.viii / Chapter CHAPTER 1 --- Introduction and Background / Chapter 1.1 --- Methods of Gene Delivery --- p.1 / Chapter 1.1.1 --- Viral Delivery Systems --- p.2 / Chapter 1.1.2 --- Non-Viral Delivery Systems --- p.3 / Chapter 1.2 --- The Gene-delivery Problems --- p.7 / Chapter 1.2.1 --- Extracellular Barriers --- p.8 / Chapter 1.2.2 --- Intracellular Barriers --- p.10 / Chapter 1.3 --- Polymer-Mediated Systems for Gene Delivery --- p.13 / Chapter 1.3.1 --- Polyethylenimine (PEI)-Based Vectors --- p.13 / Chapter 1.3.2 --- Cyclodextrin-Based Vectors --- p.15 / Chapter 1.4 --- Objective and Main Achievements --- p.16 / Chapter 1.5 --- References --- p.18 / Chapter CHAPTER 2 --- Effect of Free Polyethylenimine-Mediated Polycations on Gene Delivery: Fundamentals and Vital Factors / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Experimental Section --- p.25 / Chapter 2.3 --- Results and Discussions --- p.29 / Chapter 2.3.1 --- Fundamentals --- p.29 / Chapter 2.3.2 --- Vital Factors for the Efficacy of Free Chains --- p.37 / Chapter 2.4 --- Conclusions --- p.42 / Chapter 2.5 --- References --- p.42 / Chapter CHAPTER 3 --- Effect of Free Polyethylenimine-Mediated Polycations on Gene Delivery: Mechanistic Study / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Experimental Sections --- p.46 / Chapter 3.3 --- Results and Discussion / Chapter 3.3.1 --- Potential Effect of Free PEI Chains on Cellular Uptake --- p.49 / Chapter 3.3.2 --- Potential Effect of Free PEI Chains on Endolysosomal Release --- p.51 / Chapter 3.3.3 --- Exploration on Proton Sponge Hypothesis --- p.53 / Chapter 3.3.4 --- Interactions of PEI-based Polycations and Phospholipid Membranes --- p.55 / Chapter 3.4 --- Conclusions --- p.61 / Chapter 3.5 --- References --- p.62

Diseases of the liver have a large impact on human health. Genetic disorders, metabolic disorders, alcoholism, cancer, or infections can all impair liver function. If serious enough, a liver transplant may be necessary, a major surgical procedure which requires life-long immune suppression and relies on the availability of donor livers. Gene therapy is being intensively studied as a potential method to treat many disorders, including disorders of the liver. While viral gene therapy has seen some success, possible side effects make it risky, so nonviral gene delivery vectors are being developed. Unfortunately, these nonviral vectors do not yet have the efficiency of the viral vectors. Nonviral gene delivery vectors face many chges in vivo. The vectors must protect DNA from nucleases while it moves through the bloodstream, they must avoid nonspecific uptake, they must be enter the correct cells, and must enter the nucleus before the DNA can be expressed. If any step of this process fails, there will be very little, if any, expression, and it may be impossible to determine what went wrong. One impediment to nonviral gene delivery research is the transition from in vitro studies to in vivo studies. The cancer derived cell lines most often used for in vitro transfections are rapidly dividing, which makes nuclear entry much easier than in the whole animal. While primary cells would be a more accurate model of the in vivo environment, the number of cells that can be obtained from tissues is small, and primary cells usually cannot be cultured for long. This limits the number of experiments that can be done with each preparation of cells. To overcome this, we have miniaturized transfection assays, including the transfection of mouse primary hepatocytes with luciferase in 384 well plates. Because fewer cells are needed, more experiments can be performed with each liver preparation. Another issue introduced by the differences between in vitro and in vivo research is circulatory stability. In vitro, large particles with strong positive charges are desired, because they sink down onto the cells and are attracted to the negatively charged cellular membranes. However, in vivo these particles will aggregate serum proteins and become lodged in narrow capillary beds in the lungs or other organs, often causing toxicity. While this behavior can usually be overcome through PEGylation, improving a particle's circulatory half-life will still improve its chances of finding the correct target. Scavenger receptors found on liver nonparenchymal cells are very efficient at removing negatively charged particles from the bloodstream. We have shown that dosing large amounts of PEGylated polyacridine DNA polyplex can saturate the scavenger receptors and improve circulatory half-life. We have also shown that large doses of PEGylated peptide with or without acridine groups can inhibit scavenger receptor uptake through the formation of peptide-protein nanoparticles. By inhibiting scavenger receptor uptake, DNA can be successfully hydrodynamically stimulated at times up to 12 hours post-delivery, demonstrating a longer circulatory half-life and suggesting a mechanism to explain how delayed hydrodynamic stimulation can achieve full level gene expression in the liver after the DNA has had time to circulate throughout the whole animal. Once a nonviral vector finds its target cell, it must still enter the cell through endocytosis and then escape the endosome before it becomes digested in the lysosome. Before the DNA cargo can be expressed, it must enter the nucleus. Nuclear entry in nondividing cells is a major barrier to efficient gene delivery. One method to over come this barrier is to avoid the need for nuclear entry altogether by delivering mRNA instead of DNA. mRNA can produce protein in the cytoplasm by finding a ribosome and initiating translation. However, it is even less stable in the bloodstream than DNA. We have produced an mRNA construct capable of high-level expression in the liver through hydrodynamic delivery. The PEGylated polyacridine peptides used to protect DNA were applied to mRNA and shown to enhance expression, allowing a 1μg dose of mRNA peptide polyplex to produce higher expression than an equal dose of DNA. The peptides were also shown to provide some protection against nuclease digestion in serum. This suggests that efficient, if transient, protein expression can be achieved through peptide protected mRNA delivery. However, DNA delivery is still desired for longer term expression, and the nuclear entry of DNA is still a problem. In an effort to help facilitate nuclear entry, the membrane disrupting enzyme phospholipase A2 was modified in several ways. The enzyme was conjugated with DNA binding peptides, nuclear localization peptides, and hepatocyte targeting oligosaccharides. Additionally, mutant forms of the enzyme were prepared in bacterial expression systems to achieve site-specific conjugation. Unfortunately, none of these efforts produced a useful tool for nuclear entry. The research presented in this thesis represents some progress toward the goal of nonviral gene delivery to the liver. Hopefully, some of this work will be useful in the development of new treatments and therapies to improve human health.