Sadhna Joshi-Sukhwal

I completed my B.Sc., M.Sc., Ph.D. and D.Sc. from Université Paris Diderot, France. I then joined Allelix Biopharmaceuticals as a Senior Research Scientist and Principle Investigator on AIDS and Immune Regulation. In 1989, I joined the University of Toronto as Associate Professor in the Department of Molecular Genetics. In 2013, I received Pride of India Award and Professional Female of the Year Award from Indo-Canada Chamber of Commerce.

Research Synopsis

Research in my laboratory has been focused on the development of genetic strategies for HIV prevention and treatment. To treat HIV-infected individuals, we are developing gene therapy whereby patients own blood cells will be genetically modified to secrete antiviral proteins that would inhibit HIV infection of target cells for life.

HIV infection has become a chronic disease in highly developed countries and represents a severe burden on the patients and the health care system. Although antiretroviral drug therapy effectively suppresses HIV replication, life-long adherence to the treatment plan is required due to latent viral reservoirs that are established early in infection and are not eliminated.

Gene therapy using autologous hematopoietic stem/progenitor cells (HSPCs) offers a promising alternative as a one-time gene modification that could allow patients to produce their own antivirals for life. A number of intracellular gene therapy strategies that target HIV or CCR5 co-receptor were designed by us and others. In all cases, inhibition of HIV replication was restricted to gene-modified cells, leaving unmodified target cells vulnerable to infection. The unmodified cells will always be produced as only a fraction of HSPCs are genetically modified and there is no myeloablation.

To protect both gene-modified and unmodified target cells from becoming infected, we are developing an extracellular gene therapy strategy using secreted antiviral proteins (AVPs) that inactivate R5 and X4 HIV. We hypothesize that (a) continuous secretion of AVPs from all hematopoietic cell lineages, differentiated from the gene-modified HSPCs, will lead to therapeutic concentrations of AVPs in the blood and at the sites of HIV replication, and (b) R5 and X4 HIV inactivation will result in a systemic control of HIV infection in the absence of antiretroviral therapy, without the need to eliminate the viral reservoir.

Soluble CD4, a truncated version of the CD4 receptor, binds to R5 and X4 HIV Env glycoproteins. CD4/sCD4 binding to Env induces conformational changes that expose the co-receptor-binding site and the heptad repeat region involved in the fusion of viral and cellular membranes.

To induce these changes and allow binding to the two exposed epitopes, we fused sCD4 to a single-chain antibody 17b that targets the CCR5 and CXCR5 co-receptor-binding site or to a fusion inhibitor T45 that targets the heptad repeats.

We designed lentiviral vectors for the expression of sCD4 and the two secreted bifunctional AVPs, sCD4-scFv17b and sCD4-FIT45. Gene-modified producer cells secreted significant quantities of the AVPs that inhibited infection by the X4 and R5 HIV as well as the primary isolates.

Furthermore, continuous expression of AVPs inhibited HIV infection of peripheral blood mononuclear cells by cell-free virus as well as from infected cells. We have further optimized these vectors and conditions leading to optimal AVP secretion. Transduction of HSPCs with the optimized sinEFa-sCD4 led to high levels of gene marking (25-30%) and expression of sCD4 (1 µg/ml).

To test our approach in vivo, unmodified and gene-modified HSPCs expressing sCD4 were injected into NOD/SCID/γcnull (NSG) mice. Humanized mice supported multi-lineage differentiation from human gene-modified and unmodified HSPCs. No major differences between lineage reconstitution by gene-modified and unmodified cells were evident. Upon HIV challenge, humanized mice secreting sCD4 demonstrated a clear reduction of viral load over time and higher levels of CD4+ T cells in the peripheral blood and tissues compared to control mice.

In summary, we have shown that bifunctional AVPs can protect gene-modified and unmodified target cells in vitro against R5 and X4 HIV infections. Furthermore, we have demonstrated the feasibility of our approach in controlling HIV replication in vivo via HSPC gene therapy using lentiviral vectors expressing sCD4. We are now comparing sCD4 with each of the two bifunctional AVPs, sCD4-scFv17b and sCD4-FIT45 that target multiple steps of HIV entry.

This research could lead to an alternative therapy that may be used alone or as part of combination approach to treat and functionally cure HIV-infected individuals. If our bifunctional AVPs control HIV infection in humanized mice, we will test our strategy in a nonhuman primate (e.g. rhesus macaques challenged with SHIV) and then design clinical trials to evaluate the safety and efficacy of our approach in humans.

Genetic therapies for HIV treatment and prevention:

To treat HIV-infected individuals, we are developing gene therapy whereby our own blood cells will be genetically modified to secrete antiviral proteins that inhibit HIV infection of target cells. As gene therapy requires a one-time procedure with a life-time protection, it is cheaper than having to take antiviral drugs for life.

To prevent HIV transmission, strains of Lactobacillus that colonize the vagina and gastrointestinal tract are genetically modified to express antiviral proteins. As Lactobacillus is used to make yogurt, the engineered strains could be propagated and delivered orally. This would represent the most affordable, accessible, nutritious, safe and easy-to-use preventive measure for blocking HIV transmission.

Recent Publications

List of Selected Publications

Nazari R, Ma XZ & Joshi S (2008). Inhibition of HIV-1 entry using vectors expressing a multimeric hammerhead ribozyme targeting the CCR5 mRNA. J Gen Virol 89:2252-2261

Nazari R & Joshi S (2008). Exploring the potential of group II introns to inactivate HIV-1. J Gen Virol, 89: 2605-2610.

Nazari R & Joshi S (2009). HIV-1 gene therapy at pre-integration and provirus DNA levels. Curr Gene Ther 9: 20-25.

Falkenhagen A, Chen J, Ameli M, Asad S, Read SE & Joshi S (2011). Development and testing of a novel gene therapy strategy using secreted proteins. Mol Ther 19:1378-1379.

Joshi S (2012). HIV genetic strategies. International Innovation. June issue, pp 42-44.

Falkenhagen A, Ameli M, Asad S, Read SE & Joshi S (2013). Gene therapy using a secreted single chain variable fragment targeting CCR5 to inhibit HIV infection. J Antivir Antiretrovir 5:85-91.

Falkenhagen A, Ameli M, Asad S, Read SE & Joshi S (2014). A novel gene therapy strategy using secreted multifunctional anti-HIV proteins to confer protection to gene-modified and unmodified target cells. Gene Therapy 21:175-187.