麻豆传媒

麻豆传媒 — CAMBRIDGE, Mass. (December 19, 2016)鈥擨nvestigators from Whitehead Institute, the Ragon Institute of MGH, MIT and Harvard and the Broad Institute of MIT and Harvard have used CRISPR-Cas9 gene-editing technology to identify three promising new targets for treatment of HIV infection. In their report receiving advance online publication in Nature Genetics, the research team describes how screening with CRISPR for human genes that are essential for HIV infection but not for cellular survival identified five genes 鈥� three of which had not been identified in earlier studies using RNA interference. Their method can also be used to identify therapeutic targets for other viral pathogens.

鈥淲e were surprised to find that there are so few host factors required for HIV infection given some of the previous literature,鈥� observes David M. Sabatini, Whitehead Institute Faculty Member and co-corresponding author of the Nature Genetics paper. 鈥淭he beauty of the CRISPR-based genetic screens is the clear and robust results they yield,鈥� notes Sabatini, who is also member of the Broad Institute and Professor of Biology at Massachusetts Institute of Technology.

鈥淐urrent anti-HIV medications overwhelmingly target viral proteins,鈥� says Ryan J. Park of the Ragon Institute and the Broad Institute, co-lead author of the report. 鈥淏ecause HIV mutates so rapidly, drug-resistant strains frequently emerge, particularly when patients miss doses of their medication. Developing new drugs to target human genes required for HIV infection is a promising approach to HIV therapy, with potentially fewer opportunities for the development of resistance.鈥�

Bruce Walker, director of the Ragon Institute and co-corresponding author of the Nature Genetics paper, explains, 鈥淰iruses are very small and have very few genes 鈥� HIV has only 9, while humans have more than 19,000 鈥� so viruses commandeer human genes to make essential building blocks for their replication. Our goal was to identify human genes, also called host genes, that are essential for HIV to replicate but could be eliminated without harming a human patient.鈥�

Tim Wang, a doctoral student conducting research at Whitehead Institute and the Broad Institute, and co-lead author of the report, explains, 鈥淐RISPR makes it possible to completely knock out genes at the DNA level; and our genome-wide, CRISPR-Cas9-based approach targets more than 18,500 genes, the vast majority of human protein-coding genes. Our study demonstrates how CRISPR-based screens can be applied to identify host factors critical to the survival of other viral pathogens but dispensable for host cell viability. Broad application of this method should pinpoint a novel class of potential therapeutic targets that have previously been underexplored for the treatment of infectious disease.鈥�

Co-corresponding author Nir Hacohen, an institute member at the Broad Institute and director of Cancer Immunology at Massachusetts General Hospital (MGH), adds, 鈥淎n important aspect of our study was to focus on human T cells, the primary targets of HIV, and to identify host genes with the most dramatic role in viral infection of T cells.鈥�

Previous research has identified several host dependency factors, including two proteins required for HIV to enter CD4 T cells, the primary target of the virus: the CD4 molecule itself, to which the virus binds, and CCR5, which facilitates the binding of common HIV strains. Individuals with a particular CCR5 mutation are immune to those viral strains 鈥� indeed the only individual considered cured of HIV infection received a bone marrow transplant from a donor with that CCR5 mutation 鈥� but while therapeutic CCR5 inhibitors have been developed and are in clinical use, they can cause serious side effects.

Three 2008 studies that used RNA interference (RNAi) to identify potential host dependency factors identified more than 800 possible targets; but the little overlap among the results of the studies suggested a high rate of false positive results. In addition, none of those studies was performed using the immune cells targeted by HIV, which also reduces the likelihood that the identified genes actually participate in HIV鈥檚 infection of CD4 T cells.

Whitehead Institute鈥檚 Tim Wang explains that, 鈥淩NAi suppresses but does not completely block gene expression 鈥� which could allow a targeted gene to produce enough protein to permit HIV infection 鈥� and it also can suppress expression of additional genes besides the intended target, leading to a false positive result.鈥�

Using CRISPR to screen a cell line derived from HIV-susceptible CD4 T cells identified five genes that, when inactivated, protected cells from HIV infection without affecting cellular survival. In addition to CD4 and CCR5, the screen identified genes for two enzymes 鈥� TPST2 and SLC35B2 鈥� that modify the CCR5 molecule in a way that is required for the binding of HIV. An additional gene identified through the screen was ALCAM, which is involved in cell-to-cell adhesion. When CD4 T cells are exposed to low amounts of virus, as might be seen in natural transmission, loss of ALCAM was associated with striking protection from HIV infection.

Park explains, 鈥淎LCAM is necessary for cell-to-cell adhesion in our cell line, allowing more efficient viral transfer from one cell to the next. In fact, we found that artificially inducing the aggregation of cells lacking ALCAM restored the cell-to-cell transmission of HIV. Further studies are needed to investigate whether targeting these genes would be toxic to humans. However, even if systemic inhibition has toxic effects, gene therapy approaches that selectively target these genes only in CD4 T cells or their precursors may avoid these toxicities, although it's important to note that gene therapy remains a challenging and potentially costly therapeutic approach.鈥�

Eric S. Lander, of the Broad Institute, is a co-corresponding author of the Nature Genetics paper, along with Sabatini, Hacohen, and Walker 鈥� who is also the Phillip and Susan Ragon Professor of Medicine at Harvard Medical School, a clinician in the MGH Division of Infectious Diseases and an associate member of the Broad Institute. Additional co-authors are Dylan Koundakjian, Pedro Lamothe-Molina, Blandine Monel, Wilfredo Garcia-Beltran and Alicja Piechocka-Trocha, Ragon Institute; Judd F. Hultquist, Kathrin Schumann, Alexander Marson and Nevan J. Krogan University of California, San Francisco; Haiyan Yu, Broad Institute; and Kevin M. Krupczak, a member of the Sabatini lab at Whitehead Institute when the study was conducted. The study was supported by funds from the Ragon Institute and the Howard Hughes Medical Institute.

The study, formally titled A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors, will appear via Advance Online Publication on the Nature Genetics website on 19 December 2016.

Ryan J Park1,3,20, Tim Wang3,5, 6, 20, Dylan Koundakjian1, Judd F Hultquist7,8, Pedro Lamothe-Molina1,9, Blandine Monel1,10, Kathrin Schumann11, Haiyan Yu3, Kevin M Krupzcak5, Wilfredo Garcia-Beltran1,2, Alicja Piechocka-Trocha1, Nevan J Krogan7,8, Alexander Marson11, 15, David M Sabatini3,5, 6, 16, Eric S Lander3,4,17, Nir Hacohen3,18 & Bruce D Walker1,3,10,19顎�

1Ragon Institute of Massachusetts General Hospital (MGH), Massachusetts Institute of Technology (MIT), and Harvard University, Cambridge, Massachusetts, USA

2Harvard鈥揗IT Health Sciences and Technology, Harvard Medical School, Boston, Massachusetts, USA

3Broad Institute of MIT and Harvard University, Cambridge, Massachusetts, USA

4Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

5Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA

6David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, Massachusetts, USA

7Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biosciences, QB3, University of California at San Francisco (UCSF), San Francisco, California, USA

8Gladstone Institute of Virology and Immunology, J. David Gladstone Institutes, San Francisco, California, USA

9Biological Sciences in Public Health, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA

10Howard Hughes Medical Institute, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA

11Department of Microbiology and Immunology, University of California at San Francisco, San Francisco, California, USA

12Diabetes Center, University of California at San Francisco, San Francisco, California, USA

13Department of Medicine, University of California at San Francisco, San Francisco, California, USA

14Innovative Genomics Initiative (IGI), University of California, Berkeley, Berkeley, California, USA

15UCSF Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco, San Francisco, California, USA

16Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

17Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

18Massachusetts General Hospital Cancer Center, Boston, Massachusetts, USA

19Institute of Medical Engineering and Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

The following funding acknowledgements from the authors appear at the end of the paper:This work was supported by the Howard Hughes Medical Institute (D.M.S. and B.D.W.), the National Institutes of Health (grants CA103866 (D.M.S.), F31 CA189437 (T.W.), P50 GM082250 (A.M. and N.J.K.), U19 AI106754 (J.F.H. and N.J.K.), and P01 AI090935 (N.J.K.)), the National Human Genome Research Institute (grant 2U54HG003067-10; E.S.L.), the National Science Foundation (T.W.), the MIT Whitaker Health Sciences Fund (T.W.), the UCSF Sandler Fellowship (A.M.), a gift from J. Aronov (A.M.), the UCSF MPHD T32 Training Grant (J.F.H.), and the Deutsche Forschungsgemeinschaft (grant SCHU3020/2-1; K.S.). Support was also provided by NIH-funded Centers for AIDS Research (grant P30 AI027763, UCSF Center for AIDS Research (N.J.K.) and grant P30 AI060354, Harvard University Center for AIDS Research (B.D.W.)), which are supported by the following NIH co-funding and participating Institutes and Centers: NIAID, NCI, NICHD, NHLBI, NIDA, NIMH, NIA, FIC, and OAR. D.M.S. and B.D.W. are investigators of the Howard Hughes Medical Institute. R.J.P. is a Howard Hughes Medical Institute Research Fellow.