To this day Timothy Ray Brown, otherwise known as the Berlin Patient, is the only person cured after infection with the human immunodeficiency virus (HIV). The cure came in a fortuitous form; a stem cell transplant used to treat his leukemia came from a donor with natural resistant to HIV. Soon after, researchers discovered that a mutation in C-C chemokine receptor type 5 or CCR5, a receptor which represents the entry point for HIV into T immune cells, mediated this resistance.
Many groups attempted to recapitulate this natural resistance in the lab in an attempt to bring us closer to a cure. However, the various strategies employed to disrupt the CCR5 gene to block HIV entry have suffered from low efficiency and unwanted off-target mutations. Enter genome editing via CRISPR/Cas9 technology… The innovative technology that uses a nuclease protein (Cas9) to induce double strand breaks at DNA sites targeted via specific guide RNA molecules (gRNA).
Harvard Stem Cell Institute (HSCI) researchers Chad Cowan and Derrick Rossi have employed CRISPR/Cas9 technology to disrupt specific genes in hematopoietic stem cells (HSCs) and more differentiated cells derived from a human patient – the first study to use CRISPR/Cas9 successfully in primary cells. Their great hope is to create transplantable human hematopoietic cells immune to HIV infection.
So here’s what they did:
- They utilized a dual gRNA CRISPR/Cas9 strategy to maximize gene disruption in primary human blood cells
- Employing this strategy, they specifically disrupted the CCR5 gene in CD34+ HSCs
- The modified HSCs displayed normal renewal and differentiation capacities, and can thus reconstitute the hematopoietic system whilst also being resistant to HIV infection
- The group also disrupted the B2M gene, a component of MHC class I molecules which mediate immunogenicity, in CD8+ T immune cells. This may allow the generation a ‘‘universal’’ non-immunoreactive cells for transplantation
- Used next generation sequencing (NGS) to confirm that CRISPR/Cas9 targeting was highly gene specific, with minimal “off-target” mutations
The next obvious step is to assess functionality of modified cells in animal trials, an endeavor the researchers hope to start in the near future.
While the authors are cautious, they believe that this research is translatable into human safety trials within 5 years and they also foresee the application of CRISPR/Cas technology to a multitude of other blood-based afflictions. CRISPR/Cas advances are coming thick and fast, so who can blame them for this optimistic outlook?