Matt Richardville
Biol 509
November 8, 2010
Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O, Wang N, Lee G, Bartsevich VV, Lee YL, Guschin DY, Rupniewski I, Waite AJ, Carpenito C, Carroll RG, Orange JS, Urnov FD, Rebar EJ, Ando D, Gregory PD, Riley JL, Holmes MC, June CH: Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol 2008 , 26:808- 816.
Abstract
Objective of the paper:
The purpose of this paper was to determine whether the disruption of the CCR5 transmembrane protein will confer resistance to the HIV-1 virus. It has long been understood that HIV-1 uses the CCR5 as a co-receptor with CD4 to intitiate entry into a host cell. Glycoprotein 120 (GP120) binds with high affinity to CD4 surface protein expressed on the CD4+ T cells. This causes the virus' protein envelope to undergo a conformational change which exposes, on the GP120 viral coat protein, a chemokine binding domain which may bind either CCR5 or CXCR4 chemokine trans-membrane receptors. Discovery that a naturally occurring homozygous deletion for the Δ32 gene confers, to those individuals bearing the mutation, a resistance to the HIV-1 virus has led to extensive investigation of the role that the CCR5 . Further studies demonstrate that knock down is not sufficient to confer lasting resistance against the HIV-1 virus. It was thought that a homozygous knockout of the CCR5 gene and subsequently the CCR5 trans-membrane protein might prevent the HIV-1 virus from fusing with the CD4+ T cell thereby conferring a lasting resistance to the virus.
The experiment and results:
Previously zinc-finger nucleases (ZFNs) have been used to permanently disrupt targeted genetic sequences in eukaryotes. The use of two differing ZFNs produce non-homologous ends which repair imperfectly as a result of improper pairing via a a technique called non-homologous end joining (NHEJ). It effectively creates a disruption within the targeted gene.
The researchers custom designed ZFNs based on an archive of ZFN binding domains and the known genetic sequence of the CCR5 and IL2Rγ proteins. The CCR5 ZFN-Fok1 targeted the binding domains upstream of Δ32 gene with the intention of disrupting the CCR5 protein product. The IL2Rγ ZFN targeted an unrelated region as a control.
in-vitro: GHOST-CCR5 cells are CD4+ T cells with a green fluorescent protein (GFP) reporter for HIV infection. These cells were cultured and transduced by adenoviral vectors cloned to encode for transient expression of the CCR5 and IL2Rγ ZFNs. Gene products were isolated and amplified by radioactive PCR and separated on gel by surveyor assay. There was a substantial decrease in CCR5 for the treated cells and also a marked decrease in HIV-1 infection for those cells. There was no reduction inCCR5 expression for cells with disruption at IL2Rγ.
in-vitro: PM1 cells, a CD4 line was electroporated with a small amount of CCR5 ZFN encoding plasmids. These cells were divided into two groups, one of which underwent challenge by HIV-1 particles and the other acted as a negative control devoid of the virus. At approx. 42 days the HIV-1 challenged cell population expressed a vast increase in the ratio of CCR5 disrupted cells to Wild Type cells. The HIV-1 challenged group demonstrated selection for the CCR5 mutant and the negative control demonstrated no change in its Wild Type frequency. Therefore an infection with HIV-1 increases the prevalence of the CCR5 disruption. Models lacking the viral infection do not show this effect.
in-vivo:cells were transduced ex-vivo and injected into NOG (immunodeficient) mice. Two groups of mixed male and female received either CCR5 treated CD4+ T cells or GFP transduced CD4+ T cells. Some received the HIV-1 virus via peripheral blood mononuclear cells. After a few days, those which had received the virus showed a decrease in the ratio of CD4 T cells to CD8 T cells. This is consistent with the known effects of the HIV-1 virus on CD4 T cells populations. However, after a month the mice were sacrificed and the CD4 cells analyzed. The infected mice showed a 3 fold increase in the ratio of transduced cells to Wild Type. This indicates a heritability from one CD4 T cell to its daughter cells. A separate experiment was carried out to see the effects of the disruption in NOG mice. Viral load and peripheral CD4 cell count were measured. Greater than 50% of CD4 cells remained after 50 days of infection. The viral load was decreased in 8 out of 10 transduced and infected mice.
Conclusions:
Two significant conclusions can be drawn from these experiments. Firstly, it is possible to permanently disrupt a genetic sequence using ex-plantation, adenoviral transduction, and transient ZFN expression. It is possible to see a disruption in the function of CCR5 in the above studies. It is likely that disruption of the CCR5 gene and dysfunction of the CCR5 transmembrane receptor is the mechanism of protection. Secondly, significant resistance was conferred to most of those NOG mice which received treatment by this method. CCR5 deletion appears to provide a significant and well tolerated protective effect against the HIV-1 virus in mice. This strategy, in combination with other HIV-1 treatments, may provide much more comprehensive protection against the HIV-1 virus. Nevertheless, there are other HIV strains for which this treatment is not specific. However, this technique has potential against those and other viruses and genetic diseases if target molecules or dysfunctional gene sequences in the host are able to be identified.