Factors affecting transduction efficiency of pseudotyped viral vectors incorporating alphaviral glycoproteins

Aditi Kesari, Purdue University

Abstract

The genome of an organism has the complete set of biochemical instructions required for sustenance of life. Mutations or abnormalities in this genome lead to genetic disorders. Currently available therapeutic options mostly focus on treating the symptoms, but not curing them. Gene therapy promises to be a curative form of medicine. In gene therapy cells carrying a defective gene are targeted and replaced with a healthy copy of that gene. The vehicles used for delivering this gene are known as vectors. Retroviruses are popularly used gene therapy/transfer vectors. However, retroviruses are limited in the range of cells they can enter and infect. The range of cells targeted by the viral vector can be either expanded or narrowed by replacing the envelope of the virus with the envelope of another virus that has the desired range of tissue tropism. Such hybrid viruses are called pseudotyped viruses. Previously, we have pseudotyped Moloney Murine Leukemia Virus (MoMuLV), a retrovirus with the envelope of Ross River Virus (RRV), an alphavirus. Here, we substituted amino-acid residues of RR-envelope glycoprotein with basic amino-acid residues. We show that this makes the pseudotyped virus utilize heparan sulfate, a ubiquitous molecule present on the surfaces of most cells, as an attachment factor. Attachment to cellular heparan sulfate helps in concentration of virus particles on the cell surface and thus enhancing their chances of cell entry, thereby increasing their transduction efficiency of this viral vector. The same affinity towards heparan sulfate, however, poses a challenge in terms of release of this pseudotyped virus from the producer cells. General principles concerning viral adaptation to the use of attachment factors and improving pseudotyped virus titers through modifying cell membrane components can be derived from these results. We also show that alphavirus-glycoprotein retroviral pseudotypes require cholesterol for entry into the cells. Furthermore, excess cholesterol in cells facilitates the entry of alphavirus-glycoprotein retroviral pseudotypes. We show that alphavirus-glycoprotein pseudotypes transduce the acid sphingomyelinase deficient (ASMase-/-) cells derived from Niemann Pick’s disease-model mice more efficiently than the cells from healthy mice (ASMase+/+). Thus alphavirus-glycoprotein pseudotypes hold a potential as suitable vectors for gene therapy/ transfer in Niemann Pick’s Disease-Type A, a genetic lipid-storage disorder.

Degree

Ph.D.

Advisors

Sanders, Purdue University.

Subject Area

Genetics|Virology

COinS