CCR5 Deletion Research Articles

Approaching the asymptote

The report in 2009 of an individual who had achieved long-term remission of HIV by transplantation of hematopoietic stem cells from a donor homozygous for a deletion in CCR5 (ccr5Δ32/ccr5Δ32 galvanised research around the world aimed at curing HIV.1 As the challenges to developing safe, effective, and scalable interventions to eradicate HIV mounted, many groups engaged in parallel efforts to control rather than to eliminate the viral reservoir. Various reports of people with sustained remission of HIV provide different models for long-term control of viral replication in the absence of antiretroviral therapy (ART), including so-called elite controllers,2 post-treatment controllers,3,4 and children with perinatally acquired HIV who initiated ART early in infancy.

Factors Influencing Immune Restoration in People Living with HIV/AIDS.

Introduction: Immune restoration is a key clinical aspect that is pursued in the care of human immunodeficiency virus (HIV)-infected patients. Despite effective antiretroviral treatment and undetectable viremia, immune recovery is often incomplete. Materials and methods: Data from 311 Caucasian patients were collected. SNP in CCR2(rs1799864), CX3CR1(rs3732378), HLAC-35(rs9264942), and CCR5(promoter, rs1799988); a 32bp deletion(Δ32) in CCR5; and HLA-B*5701 genotypes were correlated with clinical data and selected endpoints. Kaplan–Meier and Cox proportional hazards models were used to analyze the effects of genetic factors over time. Results: For HLA-B*5701, the effect on the CD4+/CD8+ >0.8 cell ratio was lost within 48 months (HR = 2.04, 95% CI: 1.04–4.03), and the effect on the CD4+ cell count >500 cells/µL was lost within 12 months (HR = 2.12, CI: 1.11–4.04). The effect of CCR2 GG on the CD4+/CD8+ >0.8 cell ratio was lost within 36 months (HR = 1.7, CI: 1.05–2.75). For CCR5 wt/Δ32, the effect on the CD4+/CD8+ >1.0 cell ratio was lost within 24 months (HR = 2.0, CI: 1.08–3.69), and the effect on the CD4+ >800 cells/µL cell count was lost within 18 months (HR = 1.98, CI: 1.14–4.73). Conclusions: Selected genetic polymorphisms, namely CCR2 GG and CCR5 Δ32, and the presence of the HLA-B*5701 allele positively influenced immune restoration in cART-treated patients with HIV/AIDS.

Increased Efficiency for Biallelic Mutations of the CCR5 Gene by CRISPR-Cas9 Using Multiple Guide RNAs As a Novel Therapeutic Option for Human Immunodeficiency Virus.

CCR5 is a coreceptor of human immunodeficiency virus type 1 (HIV-1). Transplantation of hematopoietic stem cells homozygous for a 32-bp deletion in CCR5 resulted in a loss of detectable HIV-1 in two patients, suggesting that genetic strategies to knockout CCR5 expression would be a promising gene therapy approach for HIV-1-infected patients. In this study, we targeted CCR5 by CRISPR-Cas9 with a single-guide (sgRNA) and observed 35% indel frequency. When we expressed hCas9 and two gRNAs, the Surveyor assay showed that Cas9-mediated cleavage was increased by 10% with two sgRNAs. Genotype analysis on individual clones showed 11 of 13 carried biallelic mutations, where 4 clones had frameshift (FS) mutations. Taken together, these results indicate that the efficiency of biallelic FS mutations and the knockout of the CCR5 necessary to prevent viral replication were significantly increased with two sgRNAs. These studies demonstrate the knockout of CCR5 and the potential for translational development.

Combination gene therapy for HIV using a conditional suicidal gene with CCR5 knockout

BackgroundGene therapy approaches using hematopoietic stem cells to generate an HIV resistant immune system have been shown to be successful. The deletion of HIV co-receptor CCR5 remains a viable strategy although co-receptor switching to CXCR4 remains a major pitfall. To overcome this, we designed a dual gene therapy strategy that incorporates a conditional suicide gene and CCR5 knockout (KO) to overcome the limitations of CCR5 KO alone.MethodsA two-vector system was designed that included an integrating lentiviral vector that expresses a HIV Tat dependent Thymidine Kinase mutant SR39 (TK-SR39) and GFP reporter gene. The second non-integrating lentiviral (NIL) vector expresses a CCR5gRNA-CRISPR/Cas9 cassette and HIV Tat protein.ResultsTransduction of cells sequentially with the integrating followed by the NIL vector allows for insertion of the conditional suicide gene, KO of CCR5 and transient expression of GFP to enrich the modified cells. We used this strategy to modify TZM cells and generate a cell line that was resistant to CCR5 tropic viruses while permitting infection of CXCR4 tropic viruses which could be controlled via treatment with Ganciclovir.ConclusionsOur study demonstrates proof of principle that a combination gene therapy for HIV is a viable strategy and can overcome the limitation of editing CCR5 gene alone.

Targeting inhibition of CCR5 on improving obesity-associated insulin resistance and impairment of pancreatic insulin secretion in high fat-fed rodent models

Obesity is closely linked with type 2 diabetes and the effective therapies on obesity-associated diabetes are under development. The aim of this study was undertaken to investigate whether the inhibition of the augmented CCR5-mediated signaling could be a common target for treatment of obesity-associated insulin resistance and impairment of pancreatic insulin secretion in high-fat diet (HFD) fed rats and CCR5 knockout mice and also in isolated islets and RIN-m5F cells. Conducted with SD rats, HFD-induced body weight gain was significantly decreased in those combined with Maraviroc treatment, but food intake remained similar compared to control. Maraviroc also significantly improved the impaired oral glucose tolerance test (OGTT). As compared with wild-type mice, CCR5 deletion significantly attenuated the HFD-induced increases in glucose area under curve of OGTT and the value of HOMA-IR as well as plasma lipid profile. It also reversed the HFD-suppressed gene expressions of GLUT4 and IRS-1 in adipose tissue. On the other hand, the HFD-associated islet macrophage and T-cell infiltration were significantly decreased in CCR5 KO mice. H2O2 significantly suppressed glucose-stimulated insulin secretion (GSIS) is isolated islets, which were significantly reversed in those cotreated with CCR5 mAb. H2O2 failed to change GSIS in those of CCR5 KO mice. The palmitate-induced reactive oxygen species production was significantly decreased in those cotreated with CCR5 antagonist in RIN-m5F cells. Collectively, it is suggested that targeting inhibition of the CCR5 mediated inflammatory pathway could not only improve obesity-associated insulin resistance but also directly alleviate pancreatic β-cell dysfunction.

CRISPR in HIV: Dangers of CCR5 Deletion

CRISPR in HIV: Dangers of CCR5 Deletion

Lack of Association Between the CCR5-delta32 Polymorphism and Neurodegenerative Disorders.

Recent studies have suggested that diminished Ccr5 functioning has an effect on synaptic plasticity and hippocampal memory in mouse models. CCR5-delta32, a 32-bp frameshift deletion in human CCR5 encoding a nonfunctional receptor, has been reported to have a protective effect against human immunodeficiency virus infection but its role as a modifier of neurodegenerative disease has been minimally explored. We investigated whether the CCR5-delta32 polymorphism could have an effect in the context of human neurodegenerative diseases. We examined the frequency of the CCR5-delta32 polymorphism in a large and well-characterized cohort including 1425 patients with neurodegenerative dementias and 2032 controls. We did not observe a significant association between the CCR5-delta32 polymorphism and any of the neurodegenerative diseases screened in this study. However, we observed an earlier age of onset among neurodegenerative disease patients carrying the CCR5-delta32 allele. Although our findings were inconclusive, the earlier age of onset observed among neurodegenerative disease patients carrying the CCR5-delta32 allele suggests that the deletion may have a detrimental effect in the context of neurodegeneration.

CCR5 Signaling Promotes Murine and Human Hematopoietic Regeneration following Ionizing Radiation.

SummaryHematopoietic stem and progenitor cells (HSPCs) depend on regulatory cytokines from the marrow microenvironment. From an unbiased cytokine screen of murine marrow supernatants, we identified C-C motif chemokine ligand 5 (CCL5) as an endothelial cell-secreted hematopoietic growth factor. Following treatment with CCL5, hematopoietic regeneration is accelerated and survival is prolonged after radiation. In mice with deletion of Ccr5, hematopoietic regeneration is delayed compared to control mice. Deletion of Ccr5 specifically in hematopoietic cells was sufficient to delay regeneration, while the deletion of Ccr5 in stromal/endothelial cells was not. Mechanistically, CCL5 promotes hematopoietic cell cycling and cell survival. Like murine hematopoietic cells, human hematopoietic cells (cord blood, healthy marrow, and peripheral blood) increase CCR5 expression after radiation exposure to promote cell survival. These data establish that CCL5 and CCR5 signaling play critical roles in hematopoietic regeneration and could serve as therapeutic targets to shorten the duration of myelosuppression.

Chemokine Receptor Redundancy and Specificity Are Context Dependent

SummaryCurrently, we lack an understanding of the individual and combinatorial roles for chemokine receptors in the inflammatory process. We report studies on mice with a compound deletion of Ccr1, Ccr2, Ccr3, and Ccr5, which together control monocytic and eosinophilic recruitment to resting and inflamed sites. Analysis of resting tissues from these mice, and mice deficient in each individual receptor, provides clear evidence for redundant use of these receptors in establishing tissue-resident monocytic cell populations. In contrast, analysis of cellular recruitment to inflamed sites provides evidence of specificity of receptor use for distinct leukocyte subtypes and no indication of comprehensive redundancy. We find no evidence of involvement of any of these receptors in the recruitment of neutrophils or lymphocytes to resting or acutely inflamed tissues. Our data shed important light on combinatorial inflammatory chemokine receptor function and highlight Ccr2 as the primary driver of myelomonocytic cell recruitment in acutely inflamed contexts.

British Society for Gene and Cell Therapy Annual Conference and Joint UK Regenerative Medicine Platform Meeting Royal Welsh College of Music & Drama Cardiff, Wales, United Kingdom Wednesday April 19-Friday April 21, 2017 Conference Abstracts.

British Society for Gene and Cell Therapy Annual Conference and Joint UK Regenerative Medicine Platform Meeting Royal Welsh College of Music & Drama Cardiff, Wales, United Kingdom Wednesday April 19-Friday April 21, 2017 Conference Abstracts.

OR28 Advances in molecular typing of abo/rhd and ccr5Δ32 mutation

Aim ABO/RhD blood group matching is considered an important factor in hematopoietic stem cell transplantation (HSCT) since ABO incompatibility may affect the engraftment and/or ABO expressed on recipient endothelial cells may represent targets for graft versus host disease. The CCR5 deletion (CCR5Δ32) is associated with HIV resistance and provides a therapeutic strategy for eradicating HIV infection in HIV+ patients undergoing HSCT. Currently, HLA typing of NMDP donors is performed on DNA isolated from buccal swabs. The goal of this study was to develop molecular typing methods for ABO/RhD and CCR5Δ32 for HSCT registry donors. Methods 96 reference buccal swab samples were obtained from NMDP. The SSP typing for ABO/RhD and CCR5Δ32 were performed using a multiplex PCR (One Lambda) and the sizes of the amplicons were detected on 3730xl DNA Analyzer and interpreted with Gene Mapper 5. ABO typing was performed by NGS using in-house primers on Illumina MiSeq system and the results were analyzed using Galaxy (UCLA). Results For ABO blood group typing, both SSP and NGS typing methods yielded 100% concordance with reference in all major blood groups (A = 29.2%, B = 20.8%, AB = 20.8%, O = 29.2% respectively) and differentiated A1 and A2 subtypes. NGS typing identified a total of 4 A alleles, 1 B allele, and 10 O alleles with 7 novel mutations. Two mutations occurred in B101 (A930G) and O02 (G1105A). We discerned 7 O alleles without the nt261G deletion which included 5 O03 and 2 O49 alleles. O03 and O49 alleles have G802A compared to A101 which presumably inactivates the enzyme by altering its sugar-binging sites. In addition, the G1096A novel mutation was detected in all 5 O03 alleles. 100% concordance was achieved for RhD typing with 23 (24%) RhD- homozygous samples detected. In this cohort, no CCR5Δ32 double deletion was detected, but 13 CCR5Δ32/CCR5 heterozygous were found (13.5%). The CCR5Δ32 typing was confirmed by in-house SSP with 100% concordance. Conclusion We demonstrate the ability to perform accurate molecular tying of blood group ABO/RhD and CCR5Δ32. Successful molecular typing of registry donors for ABO/RhD and CCR5Δ32 may reduce donor search time and time to transplant. In addition, the ability to identify novel/rare alleles can fill in gaps in knowledge of the existing blood antigen registry and further improve the HSCT outcome.

P045 Validation of BLDType for ABO/RHD/CCR5 typing of registry donors

Aim To validate the BLDType Multiplex Typing Kit (ThermoFisher) for genotyping hematopoietic stem cell (HSC) registry donors for ABO, RHD and CCR5 using Fragment Analysis (FA). Methods Ninety-one archived genomic DNA samples (gDNAs) that had prior serology and/or ABO, RHD or CCR5 genotyping were typed using BLDType which employs fragment analysis using ABI 3130XL and Genemapper software 5 to distinguish A 1 , A 2 , B, O 1 , O 2 and O 3 alleles, detects deletion of RHD and CCR5 Δ 32. Fifty were typed for ABO and CCR5 alleles by restriction fragment length polymorphism (RFLP) and sequence-specific primer (SSP) PCR assays respectively. Twelve were genotyped for CCR5 by SSP and standard tube or automated red blood cell agglutination methods were used for ABO and RhD typing. Concordance rates were calculated for each sample subset as follows: 79 specimens with ABO and RhD phenotyping and 62 specimens with ABO by RFLP. Results and discussion Concordance between RhD serology and FA was 100% (79/79). Concordance between ABO serology and FA was 96% (76/79). The concordance between ABO RFLP and FA was 97% (60/62). The concordance between CCR5 genotyping by FA and SSP methods was 100% (91/91). The discrepancies encountered in this study were limited to ABO and were due to lack of serologic subtyping to distinguish A 1 from A 2 (N = 3) and a limitation of the RFLP which does not detect O 3 alleles (N = 2) or cannot resolve unusual banding patterns. The BLDType technique is user friendly and easy to implement. FA of 96 gDNAs can be completed in 8 h, and if automated and performed on a 3730XL, more than 1000 gDNAs can be tested per day. Conclusions The validation data presented here demonstrates that BLDType is a highly sensitive and specific typing tool. Incorporating BLDType along with HLA typing for registry donors would expedite the selection processes for both HLA-matched and ABO-compatible donors simultaneously.

CCR5Δ32 POLYMORPHISM NOT DETECTED IN HIV PATIENTS IN VOLUNTARY COUNSELING AND TESTING MOEWARDI GENERAL HOSPITAL SURAKARTA, INDONESIA

<p>Background: The CCR5.32 polymorphism (a naturally occurring 32-bp deletion in CCR5) influences the ability of HIV-1 to infect the target cells. Homozygosity for CCR5.32 prevents infection of HIV-1 R5 strain, while the heterozygous is associated with lower plasma viral load and delayed progression to acquired immune deficiency syndrome (AIDS). However, there is no report about the presentation of CCR5.32 polymorphisms in Indonesian HIV patients. The aim of this study is to detect CCR5.32 polymorphisms in Indonesian HIV patients, especially in Voluntary Counseling and Testing Moewardi General Hospital Surakarta, Indonesia. In an ongoing molecular epidemiology study of blood borne virus, 154 HIV patients in Moewardi General Hospital Surakarta were used for the study. The blood samples were collected during November – December 2011. The blood samples were aliquoted and fractionated. The DNA was extracted from all blood samples, and subjected for the PCR assay to detect the presentation of CCR5.32 polymorphisms. Internal amplification control was included in all assays. PCR products were analyzed in 3% agarose. The results showed that CCR5.32 polymorphism was not detected in all blood samples. So it can be concluded that all patients in this study had the CCR5 wild type.</p><p><br /><strong>Keywords</strong>: CCR5.32, HIV, Indonesia.</p>

West Nile Encephalitis in a previously healthy child: Evaluation for CCR5 Chemokine receptor mutation

Neuroinvasive disease seldom follows infection with West Nile virus, but is particularly rare in children. Most reported cases of West Nile virus encephalitis have occurred in older adults or the immunocompromised. Although individuals who are homozygous for a 32 base pair deletion in the chemokine receptor CCR5 have been shown to be resistant to infection with HIV-1, they have been reported to have in increased risk of developing severe disease following West Nile virus infection. Analysis of the presence of the CCR5 deletion has not been previously examined in children with West Nile neuroinvasive disease. We present a case of West Nile encephalitis in a previously healthy young child whom we evaluated for the presence of the CCR5Δ32 mutation.

Hypothesis about the protective role of ccr5delta32 mutations in juvenile idiopathic arthritis: fiction or reality?

It is suspected that the prevalence in different ethnic groups of HLA-genotype and of mutation CCR5delta32 - factors which alter adhesion of protein CCR5 - are the causes of different prevalence of juvenile idiopathic arthritis in different ethnic populations. Prerequisites to the fact that the mutation CCR5delta32 may have importance in determining susceptibility to the disease were the observations showing that CCR5 deletion polymorphism reveals a population and geographic diversity in addition to ethnic specificity. But reports on the role of gene deletion in the CCR5 chemokine receptor susceptibility to JIA rather contradictory. 234 DNA samples of patients with systemic JIA (soJIA) were ana-lyzed. The diagnosis was made according to the ILAR criteria. DNA was isolated using QIAamp Mini Kit (QIAGEN) according to the protocol provided. Our results didn’t reveal any differences in prevalence of mutation in patients with soJIA, in patients with soJIA + macrophage activation syndrome and in total population. Our results do not support the idea of protective role of the muta-tion CCR5delta32 against soJIA, which conclusion can be explained also by probable association of soJIA with HLA-genotype or other factors of ethnicity. At the same time, it can be considered as an additional evidence of expediency of soJIA being an original disease different from the rest of JIA group of diseases.

Gene Editing of CCR5 in Hematopoietic Stem Cells in a Nonhuman Primate Model of HIV/AIDS

Background: Hematopoietic stem cell (HSC) transplantation remains the only clinically observed path to functional cure of HIV infection. To better understand the mechanism of HSC-driven HIV control, and apply this therapy to a greater number of patients, we have developed a model of combination antiretroviral therapy (cART)-suppressed HIV infection in the pigtailed macaque, applicable to both gene therapy- and allogeneic transplant-based cure strategies. Following transplantation of HIV-resistant, autologous cells into conditioned animals, we are evaluating the extent to which protected cell progeny impede infection by SIV/HIV (SHIV) chimeric virus in vivo.Methods: Animals are challenged with SHIV virus containing an HIV envelope, after which a 3-drug cART regimen is initiated. Autologous HSCs are engineered to resist infection through targeted disruption of the CCR5 genetic locus using Zinc Finger Nucleases (ZFNs). Engraftment, persistence, and SHIV response of these autologous stem cells, and stem cell-derived lymphoid and myeloid cells, are measured in vivo.Results: SHIV infection in the pigtailed macaque model results in sustained viremia with consequent reduction in CD4+ T cells. Moreover, administration of three-drug cART leads to rapid and durable suppression of plasma viremia to <30 copies/mL plasma - suggesting that this model recapitulates key features of HIV infection and treatment in humans. CCR5 targeting experiments yield up to 60% gene disruption in CD34+ cells ex vivo, translating to approximately 5% disruption in vivo following transplant. Importantly, up to 10% of transplanted cells carry two disrupted alleles of CCR5; these cells should preferentially reconstitute CD4+ T-cell pools and other susceptible subsets following SHIV challenge. Consistent with this prediction, our preliminary data suggest that CCR5-deleted cells undergo positive selection following SHIV challenge in vivo.Conclusions: Our pigtailed macaque model of HIV infection and cART represents a promising platform for modeling functional cure strategies. Here we show that CCR5 deletion does not impair HSC engraftment or differentiation, and that CCR5-deleted cells can undergo SHIV-dependent positive selection even when present at low levels. Our model enables the evaluation of novel therapeutic approaches in the clinically relevant context of cART controlled SHIV infection - a setting of particular importance to approaches aimed at addressing the viral reservoir. DisclosuresWang:Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment.

Association of CCL5 and CCR5 Gene Polymorphisms With Periodontitis in Taiwanese

It has been suggested that genetic factors may predispose individuals to periodontal diseases. The present case-control study aims to test whether the -403 single nucleotide polymorphism of chemokine ligand 5 (CCL5-403) and the 32-bp deletion of CCR5 (CCR5Δ32) polymorphisms are associated with susceptibility to chronic and aggressive periodontitis. Taiwanese participants (N = 213) were grouped into control group (CG), generalized aggressive periodontitis (GAgP), or chronic periodontitis (CP) groups. DNA samples were obtained from peripheral blood. CCL5-403, evaluated by polymerase chain reaction-restriction fragment length polymorphism, and CCR5Δ32, evaluated by polymerase chain reaction, were compared among the three groups. There was a significant association between type of periodontitis and having allele A or G in the CCL5-403 polymorphism. GAgP patients were 3.7 times more likely than CP patients and 2.0 times more likely than CG patients to have allele A, instead of allele G, in CCL5-403. GAgP patients were 3.1 times more likely than CG patients to have AG versus GG genotype. GAgP patients were also 5.0 and 19.8 times more likely than CP patients to have AG and AA genotypes, respectively, compared to GG. For the CCR5Δ32 polymorphism, no association was found between the type of periodontitis and having different genotype or allele distributions among GAgP, CP, or CG patients. The single nucleotide polymorphism of CCL5-403 G substitution by A may play a role in AgP; however, the CCR5Δ32 polymorphism may not.

Seasonal trends of heart failure hospitalizations in the United States: A national perspective from 2000 to 2011

[1] Muntinghe FL, Verduijn M, Zuurman MW, et al. CCR5 deletion protects against inflammation-associated mortality in dialysis patients. J Am Soc Nephrol Jul 2009;20(7):1641–9. [2] Hutter G, Nowak D, Mossner M, et al. Long-term control of HIV by CCR5 Delta32/ Delta32 stem-cell transplantation. N Engl J Med Feb 12 2009;360(7):692–8. [3] Afzal Ali R, Kiechl Stefan, Daryani Yousef P, et al. Common CCR5-del32 frameshift mutation associated with serum levels of inflammatory markers and cardiovascular disease risk in the Bruneck population. Stroke Jul 2008;39(7):1972–8. [4] Kuhl U, Pauschinger M, Noutsias M, et al. Viral persistence in the myocardium is associated with progressive cardiac dysfunction. Circulation Sep 27 2005;112(13):1965–70. [5] Rossol M, Pierer M, Arnold S, et al. Negative association of the chemokine receptor CCR5 d32 polymorphism with systemic inflammatory response, extra-articular symptoms and joint erosion in rheumatoid arthritis. Arthritis Res Ther 2009;11(3): R91. [6] Muntinghe FL, Gross S, Bakker SJ, et al. CCR5Delta32 genotype is associated with outcome in type 2 diabetes mellitus. Diabetes Res Clin Pract Nov 2009;86(2):140–5.

HIV Voluntary Counseling and Testing of Couples During Maternal Labor and Delivery

Women in Brazil are routinely tested for HIV-1 during pregnancy with rapid testing repeated during labor in some settings. Partner testing is not routinely offered. The peripartum period provides opportunity for HIV testing of couples. A cross-sectional study was conducted in a large public hospital in southern Brazil. HIV rapid testing was offered to all pregnant women in labor. Male partners of women who consented to partner inclusion were offered testing. Within HIV-serodiscordant couples, HIV-negative individuals were evaluated for the delta-32 base-pair CCR5 deletion allele. From February to September 2009, 2888 women delivered, with 1729 eligible women approached for study participation; 1648 (95%) HIV-negative women consented to partner testing and 66% of partners accepted testing. Seven HIV-infected men (0.6%) with no prior diagnosis were identified. Testing strategies uncovered 7 additional serodiscordant couples, 4 HIV-infected women diagnosed at delivery, and 3 HIV-infected men who had not disclosed their status to their partners, for a total serodiscordance rate of 1.3% in 1101 couples. No cases of acute maternal or infant infection were noted. No delta-32 base-pair deletions were identified in 14 HIV-negative partners in serodiscordant relationships. Parameters associated with increased acceptance of partner testing included higher income (P = 0.003), education (P < 0.0001), stable relationships of longer duration (P = 0.001), and female support of partner testing (P < 0.0001). Testing of couples at the time of labor and delivery is a feasible public health strategy in areas of moderate-to-high HIV prevalence, which can potentially prevent acute infections in men, women, and infants.

Genomic Editing of the HIV-1 Coreceptor CCR5 in Adult Hematopoietic Stem and Progenitor Cells Using Zinc Finger Nucleases

The HIV-1 coreceptor CCR5 is a validated target for HIV/AIDS therapy. The apparent elimination of HIV-1 in a patient treated with an allogeneic stem cell transplant homozygous for a naturally occurring CCR5 deletion mutation (CCR5Δ32/Δ32) supports the concept that a single dose of HIV-resistant hematopoietic stem cells can provide disease protection. Given the low frequency of naturally occurring CCR5Δ32/Δ32 donors, we reasoned that engineered autologous CD34+ hematopoietic stem/progenitor cells (HSPCs) could be used for AIDS therapy. We evaluated disruption of CCR5 gene expression in HSPCs isolated from granulocyte colony-stimulating factor (CSF)-mobilized adult blood using a recombinant adenoviral vector encoding a CCR5-specific pair of zinc finger nucleases (CCR5-ZFN). Our results demonstrate that CCR5-ZFN RNA and protein expression from the adenoviral vector is enhanced by pretreatment of HSPC with protein kinase C (PKC) activators resulting in >25% CCR5 gene disruption and that activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway is responsible for this activity. Importantly, using an optimized dose of PKC activator and adenoviral vector we could generate CCR5-modified HSPCs which engraft in a humanized mouse model (albeit at a reduced level) and support multilineage differentiation in vitro and in vivo. Together, these data establish the basis for improved approaches exploiting adenoviral vector delivery in the modification of HSPCs.