Tailored gene therapy and HIV infection resistance


Since the development of AZT (zidovudine), the first antiretroviral drug, Highly Active Antiretroviral Therapy (HARRT) proved to be very efficient in the prolongation and improvement of the HIV-infected patients’ lives. Thanks to the combined anti-retroviral therapy, HIV is no longer a universally fatal disease, but a potentially chronic one. However, despite this success, HIV therapy is not completely effective and presents, along with toxic effects, increased risks of non-AIDS morbidity and mortality, pressing the need to develop a cure for HIV infection.


Stem cell therapy, the hope.


Several steps of the HIV life cycle can be targeted in order to stop the virus to spread in the organism, one of which would be its binding and fusion to target cells, CD4+ T cells. This step requires the virus binding to 2 molecules expressed on the T cell surface; a specific receptor CD4 and a co-receptor CCR5. If one of these 2 molecules carries a mutation in the binding site, no binding can occur sparing the cell from the infection.


A very small number of individuals (less than 1%) carry a mutation in the CCR5 gene (CCR5D32) making them naturally resistant to HIV infections. In 2009, a case report was published describing a patient (known as the Berlin patient), which has been cured from HIV infection following allogeneic hematopoietic stem cell transplantation from a homozygous CCR5D32 donor. Although bone marrow transplantation is not likely to be an option, the Berlin patient marks the first step towards cell therapy as a treatment for HIV infection.


The rarity of individuals carrying the CCR5D32 mutations makes the probability of finding a proper match for hematopoietic cells’ transplantation very difficult. The solution resides in the Induced pluripotent stem cells (also known as iPS cells), developed for the first time in 2006. John B. Gurdon and Shinya Yamanaka, Nobel price of medicine in 2012, demonstrated that with proper manipulations, differentiated cells can be reprogrammed and acquire pluripotent stem cells’ characteristics. iPS cells, once generated, can de differentiated to any kind of adult specialised cells using specific environment and proper signalling molecules’ cocktails.


Gene tailoring, the solution


Producing iPS cells from CCR5D32 individuals, differentiating them to hematopoietic cells and transplanting them into HIV infected patients may seems to be a very exciting idea, limited by the necessity of HLA class I compatibility. A solution was proposed in April 2013, when a group of researchers at the Stanford University School of Medicine discovered a novel method based on gene tailoring. Using a Zinc Finger Nuclease (ZFN, sequence specific nuclease) developed by Sangamo BioSciences in Richmond, California, Dr. Porteus and his colleagues were able to mimic the CCR5D32 mutation and transform a T cell line, making it resistant to HIV infection. Using the same ZFN, researchers at the University of Pennsylvania, Philadelphia, have successfully achieved the first application of gene therapy on 12 HIV infected patients. In the study published in the New England Journal of Medicine in March 2014, researchers extracted CD4+ T cells from the patients, modified them by removing the CCR5 gene using ZFN, and then returned them to the patient by blood transfusion. In the presence of HIV, modified CD4+ T cells survived longer than the normal ones even though the antiretroviral therapy was stopped for some patients from the cohort. Although the patients continued to be tested positive for HIV throughout the study, the results of this Phase I trial are very encouraging as they prove the technique’s  safety.


In the same line as the previous studies, Yuet Wai Kan, former President of the American Society of Haematology, and his colleagues highlighted the importance of manipulating CCR5 by gene editing, as the privilege path toward an efficient gene therapy against HIV infection. In their paper, published in July 2014 in PNAS, Kan et al. were able to produce a CCR5D32 mutation precisely matching the one naturally occurring in the HIV resistant individuals, and that by using the piggyBac Transposon, considered safer than ZFN. Although nucleases are designed to break the DNA in one spot, lentiviral or adenoviral ZFN recognise 12bp, statistically occurring once every 1,7×10e7 bp, so 177 times in the human genome, causing breaks elsewhere than the desired spot, possibly leading to yet-undiscovered deleterious effects. By applying the piggyBac technology to iPS cells, they were able to generate Monocytes/macrophages resistant to HIV infection. “The future goal to treatment is to take skin cells from patients, differentiate them into iPS cells, correct the mutations by homologous recombination, and differentiate into the hematopoietic cells and re-infuse them into the patients. Since the cells originate from the patients, there would not be immuno-rejection”, sais Prof. Kan.


While waiting for the ultimate vaccine for HIV, several gene editing experiments and trials strongly support the idea that the use of genetically modified cells is a promising approach for altering the course of HIV infection.




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