Everyone knows someone affected by cancer. It is a heterogeneous malignancy requiring different approaches depending on the source, aggressiveness, and stage of the disease. To this end, innovative cancer therapies are required to conquer the unique obstacles posed by each type of cancer. One emerging therapeutic avenue employs the use of oncolytic viruses. Oncolytic viruses are predominantly attenuated viruses that specifically replicate in cancerous cells, which often have defective anti-viral responses, while leaving normal tissue unaffected. The inability of certain cancers to counter viral infections stems from a defective interferon pathway utilized by the malignant cells for unregulated proliferation. This ingenious exploitation of the cancer’s double-edged attribute led to numerous oncolytic virus clinical trials presently culminating in an approved oncolytic virus therapy, Talimogene laherparepvec, for the treatment of advanced melanoma. Oncolytic measles virus is currently being evaluated in several pre-clinical and clinical cancer trials. This virus offers many advantages as a replicating cancer therapeutic such as an excellent safety profile, oncotropic traits, and permissiveness for enhancement via genetic engineering. Even so, further improvements of oncolytic measles virus may be required to overcome the various complexities that each type of cancer poses. Some concerns are also inherent to the use of measles virus itself, such as pre-existing neutralizing antibodies towards the virus from routine immunization. This thesis outlines three distinct projects which aim to improve oncolytic measles virus as a cancer therapeutic. Firstly, a novel pseudotyping platform for oncolytic measles virus is described as an efficient and robust system for viral envelope exchange for the purpose of evading neutralizing antibodies. Secondly, oncolytic measles virus armed with granzyme B displayed increased oncolytic and proinflammatory activity. Finally, synergizing oncolytic measles virus with viral sensitizers enhanced the replication and cancer cell killing ability of the virus in both human and murine cancer models. Each project uniquely demonstrated advances in improving oncolytic measles virus so it may surmount the current challenges facing it as a cancer therapeutic.

Lentiviral gene therapy has held great promise for treating a wide range of neurological disorders due to its ability to stably integrate into the genome of nondividing cells like neurons, in addition to dividing cells. The nervous system is a complex and highly heterogeneous system, and while a therapeutic intervention may have beneficial effects in one population of cells it may have severe side effects in another. For this reason, specific targeting of lentiviral vectors is crucial for their ultimate utility for research and clinical research use. Two different approaches for focusing the targeting of lentiviral vectors were employed in these studies. The first method involved assessing the effects of vector production strategies on the resulting virus’s tropism both in vivo and in vitro. The changes in vector transduction were determined via flow cytometry on cells in culture and immunohistochemistry following brain injections. Results from these experiments suggest that while the production conditions do impact the vectors efficacy, there is not a distinct effect on their tropism. A unique characteristic of retroviral and lentiviral vectors is their capacity for being pseudotyped, conferring a new tropism on the vector. Native tropisms are generally not specific beyond very broad cell types, which may not be sufficient for all applications. In this case, chimeric targeting molecules can provide an even more refined targeting profile compared to native pseudotypes. The second approach utilizes novel chimeric glycoproteins made from nerve growth factor and the vesicular stomatitis virus glycoprotein. These chimeras are designed to pseudotype lentiviral vectors to target nociceptive sensory neurons for a variety of disorders. While these chimeras were successfully produced as protein, they were misfolded and sequestered in the endoplasmic reticulum and therefore unavailable to produce lentivirus. While neither strategy was completely successful, they do provide interesting information for the design and creation of lentiviral vectors. This research shows that small differences in the steps followed as part of a lentivirus production protocol can greatly impact the resulting vectors efficacy. It also shows that while VSV has been used to create chimeric glycoproteins, not all targeting molecules are suitable for this purpose.

Gene therapy is the delivery of genetic material to cells for a therapeutic effect. Retroviruses are one of the most common viral vectors used for gene therapy, especially lung gene therapy. However the lung has many physical and immunological barriers to gene transfer vectors, and currently, too few cells are genetically modified for the effective treatment of lung diseases such as cystic fibrosis. One of the main reasons for low cell transduction is the lack of commonly-used receptors for gene therapy vectors on the apical surface of polarized epithelial cells. The objective of this project was to determine how to incorporate proteins into the lentiviral lipid bilayer in order to develop a recombinant retrovirus that can efficiently deliver genes to polarized epithelial cells via their apical membranes. We analyzed the process of incorporating heterologous viral and non-viral proteins into lentiviruses and determined key factors that allowed for successful protein incorporation into the lentiviral lipid bilayer. We found that lipid rafts segregated raft proteins, and for a protein to be incorporated into virus particles, it must be colocalized with lentivirus-associated rafts. When cells were treated with the cholesterol-extracting compound, methyl-beta-cyclodextrin, previously sequestered viral and non-viral raft proteins were then colocalized and non-viral proteins were incorporated into lentiviruses. We also created a lentivirus pseudotyped with envelope proteins from human parainfluenza type 3 (HPIV3), which naturally targets polarized epithelial cells of the lung. Lentiviruses were able to incorporate HPIV3 glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F), and were able to transduce polarized cells in a manner consistent with lentiviral-mediated transduction via sialated receptors for HPIV3, however titers were too low for clinical use. We increased protein expression of HN and found that while expression, envelope incorporation, and titer increased, lentiviruses still incorporated too few envelope proteins for efficient transduction. We determined low envelope incorporation rates were due to lack of interactions with Gag, and increasing active and passive interactions with Gag enhanced HN and F incorporation into lentiviruses. Overall, this research is significant because it provides insight into viral assembly and protein incorporation for the generation of pseudotyped lentiviruses for human gene transfer.

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Le transfert de gènes dans les cellules souches hématopoïétiques par des vecteurs lentiviraux s’inscrit dans les protocoles actuels de traitement par thérapie génique de plusieurs maladies monogéniques (B-thalassémie, Adrénoleucodystrophie, SCID…). De même, le transfert de gènes dans les lymphocytes T et B ouvre des perspectives tant au niveau de la thérapie génique que pour l’immunothérapie. Nous avons mis au point des vecteurs lentiviraux pseudotypés par des glycoprotéines chimérique (BaEV/TR) et mutante (BaEVRLess) du rétrovirus endogène de babouin. Nous avons montré que ces nouveaux vecteurs peuvent transduire de manière plus efficace les cellules souches hématopoïétiques stimulées et quiescentes que les vecteurs pseudotypés par la glycoprotéine du virus de la stomatite vésiculaire (VSV-G). Il en est de même pour les vecteurs développés récemment et pseudotypés par les Glycoprotéines H et F du virus de la rougeole. Nous avons aussi comparé la capacité de ces derniers vecteurs à ceux pseudotypés par les glycoprotéines BaEV/TR et BaEVRLess dans le transfert de gènes dans les lymphocytes B et T ainsi que dans l’ensemble des cellules de la lignée T. Nous sommes désormais en mesure de proposer des vecteurs adaptés au transfert de gènes à chaque étape de la différenciation des cellules CD34+ en thymocytes ainsi qu’en lymphocytes T matures. Ceci pourrait permettre de proposer de nouveaux protocoles cliniques en thérapie génique avec une co-transplantation de cellules souches génétiquement modifiées et de cellules T différenciées à partir de ces cellules. Ceci permettrait notamment de réduire les phases d’aplasie actuellement nécessaires pour la greffe de cellules souches. / Lentiviral vectors and their ability to transfer gene into hematopoietic stem cells are currently evaluated for the cure of several single-gene diseases (eg : B-thalassemia, Adrenoleucodystrophy, SCID). Likewise, gene transfer into B and T lymphocytes is of major interest in gene therapy and immunotherapy. We engineered new lentiviral vectors pseudotyped by some chimeric (BaEV/TR) and mutant (BaEVRLess) glycoproteins from the baboon endogenous retrovirus. We demonstrated that these new vectors can transduce more efficiently resting and mild stimulated hematopoietic stem cells than obtained with lentivectors pseudotyped by the glycoprotein G from the vesicular stomatitis virus (VSV-G). It is the same with the recently developed lentiviral vectors pseudotyped by the H and F glycoprotein from measles virus (H/F-LVs). We also compared the ability of the H/F-LVs with the BaEV/TR and BaEVRLess lentiviral vector pseudotype to transfer genes into B and T lymphocytes and into the whole T lineage. From now on, we are able to propose adapted vectors for gene transfer at each stage of differentiation from CD34+ cells to thymocytes and mature T cells. This could allow us to propose some new clinical protocols in gene therapy with a co-transplantation of genetically modified stem cells and their differentiated T progenitors in order to reduce the aplasia stage induced by current transplantation protocols.

Like all other viruses, a successful egress of functional particles from infected cells is a prerequisite for foamy virus (FV) spread within the host. The budding process of FVs involves steps, which are shared by other retroviruses, such as interaction of the capsid protein with components of cellular vacuolar protein sorting (Vps) machinery via late domains identified in some FV capsid proteins. Additionally, there are features of the FV budding strategy quite unique to the spumaretroviruses. This includes secretion of non-infectious subviral particles and a strict dependence on capsid-glycoprotein interaction for release of infectious virions from the cells. Virus-like particle release is not possible since FV capsid proteins lack a membrane-targeting signal. It is noteworthy that in experimental systems, the important capsid-glycoprotein interaction could be bypassed by fusing heterologous membrane-targeting signals to the capsid protein, thus enabling glycoprotein-independent egress. Aside from that, other systems have been developed to enable envelopment of FV capsids by heterologous Env proteins. In this review article, we will summarize the current knowledge on FV budding, the viral components and their domains involved as well as alternative and artificial ways to promote budding of FV particle structures, a feature important for alteration of target tissue tropism of FV-based gene transfer systems.

Like all other viruses, a successful egress of functional particles from infected cells is a prerequisite for foamy virus (FV) spread within the host. The budding process of FVs involves steps, which are shared by other retroviruses, such as interaction of the capsid protein with components of cellular vacuolar protein sorting (Vps) machinery via late domains identified in some FV capsid proteins. Additionally, there are features of the FV budding strategy quite unique to the spumaretroviruses. This includes secretion of non-infectious subviral particles and a strict dependence on capsid-glycoprotein interaction for release of infectious virions from the cells. Virus-like particle release is not possible since FV capsid proteins lack a membrane-targeting signal. It is noteworthy that in experimental systems, the important capsid-glycoprotein interaction could be bypassed by fusing heterologous membrane-targeting signals to the capsid protein, thus enabling glycoprotein-independent egress. Aside from that, other systems have been developed to enable envelopment of FV capsids by heterologous Env proteins. In this review article, we will summarize the current knowledge on FV budding, the viral components and their domains involved as well as alternative and artificial ways to promote budding of FV particle structures, a feature important for alteration of target tissue tropism of FV-based gene transfer systems.

Foamy viruses (FVs) or heterologous retroviruses pseudotyped with FV glycoprotein enable transduction of a great variety of target tissues of disparate species. Specific cellular entry receptors responsible for this exceptionally broad tropism await their identification. Though, ubiquitously expressed heparan sulfate proteoglycan (HS-PG) is known to serve as an attachment factor of FV envelope (Env)-containing virus particles, greatly enhancing target cell permissiveness. Production of high-titer, FV Env-containing retroviral vectors is strongly dependent on the use of cationic polymer-based transfection reagents like polyethyleneimine (PEI). We identified packaging cell-surface HS-PG expression to be responsible for this requirement. Efficient release of FV Env-containing virus particles necessitates neutralization of HS-PG binding sites by PEI. Remarkably, remnants of PEI in FV Env-containing vector supernatants, which are not easily removable, negatively impact target cell transduction, in particular those of myeloid and lymphoid origin. To overcome this limitation for production of FV Env-containing retrovirus supernatants, we generated 293T-based packaging cell lines devoid of HS-PG by genome engineering. This enabled, for the first, time production of inhibitor-free, high-titer FV Env-containing virus supernatants by non-cationic polymer-mediated transfection. Depending on the type of virus, produced titers were 2- to 10-fold higher compared with those obtained by PEI transfection.