Long-lived T cells for treating HIV infection

Abstract

The present invention provides a method for generating a T-cell enrichment population for treating HIV infection, an isolated enrichment population of T cells, and a method for treating an HIV-infected subject. [Solution] A method for treating an HIV-infected subject involves CD45A and CD45O(RA) int RO int This involves administering the enriched CCR5 and / or CXCR4 gene-edited CD4+ T cell population characterized by the co-expression of intermediate cell surface genes.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Application No. 62 / 875,217, filed on 17 July 2019, the subject matter of which is incorporated herein by reference in its entirety. [Background technology]

[0002] The presence of a small pool of latent infected cells is a major obstacle to the cessation of antiretroviral therapy (ART) and the eradication of human immunodeficiency virus (HIV). While ART can permanently suppress viral replication, HIV persists indefinitely, and infected individuals need to continue complex antiretroviral drug regimens for life. The ability of ART to reconstruct immune function is highly variable. A subset of individuals (up to 45%) cannot show a full recovery of CD4+ T cell counts even after years of effective ART. Impaired CD4+ T cell recovery has been associated with many host-related and HIV-related factors, including impaired thymogenesis and homeostasis. Low CD4+ T cell counts in individuals undergoing ART are associated with a high risk of cancer and other diseases, thus novel therapeutic approaches are needed to enhance the immune function of such individuals.

[0003] Studies modeling latent HIV reservoirs have shown minimal decay of total HIV DNA and integrated HIV DNA four years after ART initiation, particularly in individuals who initiated ART during the chronic phase of infection. Several mechanisms contribute to HIV persistence, including homeostatic proliferation, dysfunctional host clearance mechanisms, and "latent" infection of long-lived memory CD4+ T cells maintained, possibly by residual viral replication. Interestingly, all of these mechanisms are exacerbated in immunological nonresponders and associated with a larger HIV reservoir size. Therefore, enhancing CD4+ T cell recovery may contribute to reducing the HIV reservoir during ART.

[0004] CCR5 is one of the primary co-receptors for HIV entry. The therapeutic concept of providing a CCR5-deficient immune compartment to an HIV-infected subject involves providing CD34 from a homozygous CCR5Δ32 matched donor. + This was demonstrated in a “Berlin patient” who had not been infected with HIV since receiving an allogeneic bone marrow transplant of stem cells. While these results are promising, less invasive and more broadly applicable therapeutic strategies would be desirable. One approach is to reconstitute immune function through adoptive transfer of autologous T cells, which has worked well in other viral infections, including cytomegalovirus and Epstein-Barr virus, but has largely failed in HIV infection, partly because CD4+ T cells retain susceptibility to HIV infection. A recent trial in which adoptive transfer of zinc finger nuclease (ZFN)-mediated CCR5 gene-edited CD4 T cells (SB-728-T product) was performed in a group of HIV-infected adults showed that this infusion was safe and well-tolerated, leading to an increase in CD4+ T cell count and a decrease in HIV reservoir. [Overview of the project]

[0005] The embodiments described herein are CD45RA int CD45RO int phenotypic CD4 T cells and CD8 T cells (CD4 / CD8 T cells), CD45RA int CD45RO intLong-lived enriched populations of genetically modified and / or altered CD4 / CD8 T cells having a phenotype, and their use in the treatment of latent HIV infection in HIV-infected subjects, particularly subjects who are receiving and / or continue to receive antiretroviral therapy. A subset of CD4 / CD8 T cells has been found to have the phenotype and molecular attributes of long-lived multipotent stem cells. Similar to other known stem cell populations, this subset population has a low metabolic profile (upregulation of fatty acid metabolism and oxidative phosphorylation, and downregulation of the cell cycle pathway), retains self-renewal capacity, and can differentiate into effector cells. This subset is characterized primarily by intermediate co-expression of CD45RA and CD45RO (CD45RA int CD45RO int ). CD45RA int CD45RO int CD4 / CD8 T cells having the phenotype can also express CD95 (Fas), CD127 (IL7R), and CD27. Addition of low doses of the cytokines IL-7 and IL-15 may lead to the formation of an enriched population of CD4 / CD8 cells having the CD45RA int CD45RO int phenotype, while high doses of the cytokines IL-7 and IL-15 may lead to effector differentiation of the cells.

[0006] CD45RA int CD45RO int CD4 / CD8 T cells having the phenotype can be genetically modified such that they lack functional CCR5 and / or CXCR4 HIV co-receptors. Administration of CCR5 and / or CXCR4 gene-edited autologous CD4 / CD8 T cells having the CD45RA int CD45RO int phenotype to HIV-infected subjects can result in a sustained increase in CD4+ T cell numbers, restoration of T cell homeostasis, and a substantial reduction in the size of the HIV reservoir in the subject.

[0007] In some embodiments, CD4 / CD8 T cells may be genetically modified to lack functional CCR5 and / or CXCR4 HIV co-receptors, CD45RA int CD45RO int A method for generating a phenotypic enriched population of CD4 / CD8 T cells involves isolating T cells from a biological sample of the subject. The biological sample may include T cells from a sample such as peripheral blood mononuclear cells of the subject with HIV being treated, i.e., autologous T cells from the subject being treated. The isolated T cells may contain CD4+ T cells and / or CD8+ T cells.

[0008] CD45RA int CD45RO int A population of CD4 / CD8 T cells exhibiting the phenotype can be isolated from isolated T cells. In some embodiments, CD4 / CD8 T cells are CD45RA int CD45RO int Before isolating a population of phenotypic CD4 / CD8 T cells from isolated T cells, the CD4 / CD8 T cells may be genetically modified to lack functional CCR5 and / or CXCR4 HIV co-receptors. In another embodiment, CD45RA int CD45RO int A population of CD4 / CD8 T cells exhibiting the phenotype was isolated from isolated T cells and then subjected to CD45RA int CD45RO int A population of phenotypic CD4 / CD8 T cells may be genetically modified to lack functional CCR5 and / or CXCR4 HIV co-receptors.

[0009] In some embodiments, isolated CD4 / CD8 T cells are genetically modified by at least one of transduction, transfection, and / or electroporation to inactivate the genes encoding CCR5 and / or CXCR4 within the cell.

[0010] In some embodiments, isolated CD4 / CD8 T cells can express at least one of CD95, CD127, or CD27. In other embodiments, isolated CD4 / CD8 T cells can intermediately express 4-1BB and optionally OX40.

[0011] In other embodiments, isolated CD4 / CD8 T cells can express at least one, at least two, at least three, at least four, or at least five or more of the following: IL17RA, CD5, IL2RG, IGF2R, SLC38A1, IL7R, SLC44A2, SLC2A3, CD96, CD44, CD6, CCR4, IL4R, or SLC12A7.

[0012] In some embodiments, isolated CD4 / CD8 T cells are CD45RA int CD45RO int It can have a CD95+CD127+CD27+ phenotype. In another embodiment, isolated T cells have a CD45RA phenotype. int CD45RO int CD95+CD127+CD27+IL7R+CD44+SCL38A1+IL2RG+CD6+CD5+ may have the following phenotypes:

[0013] In other embodiments, this method may include activating isolated CD4 / CD8 T cells with an anti-CD3 antibody and / or anti-CD28 antibody before gene modification and / or isolation. The activated CD4 / CD8 T cells are then treated with CD45RA int CD45RO int They can be cultured in amounts of IL7 and IL15 effective in promoting the expansion and / or formation of enriched populations of phenotypic CD4 / CD8 T cells. Once isolated, CD4 / CD8 T cells are CD45RA int CD45RO int To maintain the phenotype, the cells can be cultured in a culture medium containing TGFβ / IL-1β.

[0014] Other embodiments described herein relate to compositions comprising an enriched population of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells produced by the method described herein. At least about 70%, at least about 75%, at least about 80%, at least 85%, at least about 90%, and at least about 95% of the enriched population of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells are CD45RA int CD45RO int The phenotype may be present. The composition or enriched T cell population can be administered to a subject having HIV infection to treat HIV infection. In some embodiments, administration of the composition or enriched T cell population to a subject having HIV can promote at least one of the following: a sustained increase in absolute CD4 cell count in the subject, restoration of HIV-specific T cell immunity, and substantial decay of the HIV reservoir. In some embodiments, the subject is receiving and / or continuing to receive antiretroviral therapy. [Brief explanation of the drawing]

[0015] [Figure 1] This flowchart illustrates a method for generating an enriched population of CD4 / CD8 T cells possessing the CD45RAintCD45ROint phenotype. [Figure 2A-F]Plots showing the decay of the HIV reservoir after SB-728-T injection correlate with the persistence of CCR5 gene-edited cells. A. Box plots with jitter overlaid showing the frequency of cells containing total HIV DNA per 10⁶ PBMCs at baseline (BL), 1 year post-injection, and 2 years post-injection. Boxes show the median, first and third quartiles, and whiskers extend to the maximum and minimum values. Individual data points are indicated in colors corresponding to different cohorts for all nine participants (cohorts 1, 2, and 3 are shown in blue, green, and red, respectively). BL values ​​for subjects 1-01 and 1-02 were entered as described in Materials and Methods. *P<0.05, Wilcoxon rank-sum test. B. Frequency of integrated HIV DNA copies per 10⁶ purified CD4+ T cells is shown at BL and 2-3 years (long-term follow-up). Participants in cohorts 1, 2, and 3 are indicated by blue, green, and red symbols, respectively. *P<0.05, Wilcoxon rank-sum test. C-D, association between changes in the frequency of PBMCs carrying total HIV DNA at long-term time points (ratio of log10 value at day 720 to day 0) and pentameric replication and magnitude expansion of marked cells at day 21 (C) and years 3-4 (D) after injection. Scatter plots and predictions from robust regression models are shown with 95% confidence intervals (shaded areas). E, representative example of biphase decay analysis of HIV DNA (participant 3-01) using Monolix, a nonlinear mixed-effects model parameter estimation software. The blue lines represent the biphase exponentially fitted lines for HIV DNA copies per 106 PBMCs (represented by red stars). These lines represent fast and slow decay, and the plateau reached after the end of the slow decay phase, respectively. The inset highlights two intersections representing the start and end of the slow phase decay. F. A representative example of the estimated total HIV DNA per 10⁶ PBMCs (red line) expected as a result of post-injection dilution (participant 3-01). The measured frequency of total HIV DNA per 10⁶ PBMCs (blue line) and the estimated frequency of total CCR5 gene-edited cells per 10⁶ PBMCs (purple line, calculated by multiplying the frequency of pentameric replicated and marked cells by 4) are also shown. [Figure 3A-D]Graphs and plots are shown illustrating the identification of a novel memory stem cell CD4+ T cell subset (CD45RAintROint cells expressing CD95) that contributes to the persistence of CCR5 gene-edited T cells and total CD4+ T cells, but contributes minimally to the CD4+ T cell reservoir. Bar graphs show the mean distribution of naive, TCM, TTM, TEM, and CD45RAintROint frequencies in CD4+ T cells at A, BL (7 days to 3 months before injection, n=9), early (14 to 28 days after injection, n=6), mid-term (4 to 7 months, n=7), and long-term (3 to 4 years, n=9) time points. *P<0.05, **P<0.01; Wilcoxon rank-sum test. B. Median frequency of pentamer replication markers per 106 cells measured in selected TCM, TTM, and TEM memory subsets at 14 days to 4 months (n=7 for all three subsets), 6 to 8 months (n=7, 7, and 6, respectively), 11 to 12 months (n=7, 7, and 6, respectively), and 3 to 4 years (n=7, 7, and 5, respectively), as well as in CD45RA+TSCM and CD45RAintROintTSCM at 9 to 10 months post-injection (n=6 and 5, respectively), 11 to 12 months (n=3 and 5, respectively), and 3 to 4 years (n=7 and 8, respectively). N / A = Not performed; limitations of cryopreserved PBMCs prevented quantification of the TSCM subset at the initial time point. C. Box plots with jitter overlaid showing the contribution of each subset to the CD4+ T cell HIV reservoir in samples from years 3–4 (n=8 due to limitations in cell availability). The boxes show the median, first and third quartiles, and the whiskers extend to the maximum and minimum values. The p-values ​​of the Wilcoxon rank-sum test are shown. D. Three-dimensional scatter plots showing the change in the frequency of PBMCs carrying total HIV DNA after injection as a function of the number of CD45RAintROintTSCM cells from years 3–4 (ratio of log10 value at day 720 to day 0), the frequency of pentameric replication in CD45RAintROintTSCM from years 3–4, and the ratio of the frequency of pentameric replication in CD45RAintROintTSCM from years 3–4 to the frequency of pentameric replication in TEM. A sparse linear multivariate model was constructed to predict reservoir decay.The multivariate regression model predicting the best reservoir decay included three features: CD45RAintROintTSCM cell count at years 3–4 (z-axis), log10 pentamer replication level in CD45RAintROintTSCM at years 3–4 (x-axis), and pentamer replication ratio in CD45RAintROintTSCM / TEM at years 3–4 (y-axis). Each dot in the scatter plot corresponds to a participant whose dot size is proportional to the 720-day / BL ratio of HIV DNA, with larger decays symbolized by smaller dot sizes. [Figure 4A-G]Tables, graphs, and plots showing CD45RAintROintTSCM demonstrate its differences from previously identified CD45RA+TSCM cells. A. Heatmap (n=7) of selected pathways significantly enriched in induced or repressed genes in CD45RAintROintTSCM compared to TEM and TCM in 3-4 year samples. The color gradient shows the GSEA normalized enrichment score (NES in the range of -4 to +5) of pathways enriched in induced or repressed genes in CD45RAintROintTSCM compared to TEM and TCM (P<0.05). Selected pathways were grouped into several biological functions: cell cycle, cell metabolism, cytokine signaling, Notch signaling, and apoptosis. B. Distribution of ZFN-mediated CCR5 mutations determined by DNA sequencing, uniquely present in CD45RAintCD45ROint CCR7+CD27+ in the SB-728-T product in a 3-4 year CD4+ T cell subset (n=5). The boxes show the median, first and third quartiles, with whiskers extending to a distance of 1.5*IQR. Outliers are indicated by dots. C. Pie charts and bar graphs showing the frequencies of IFN-γ, IL-2, and TNF-α cytokines produced in CD4+ T cell subsets 3-4 years post-infusion in response to anti-CD3 / CD28 stimulation. Responses are averaged for each cell subset (n=6). Pie charts show the proportion of cells yielding 1, 2, or 3 functions. Arcs identify cell populations positive for IL-2, IFN-γ, and TNF-α. Bar graphs show the relative frequencies of different combinations of cytokine production. D. Histograms (n=7) showing the expression of transcription factors T-bet, Eomes, RORgt, and GATA-3 in CD4+ T cell subsets 3-4 years post-infusion. *P<0.05, Wilcoxon rank-sum test. E. A multidimensional scaling (MDS) plot using Euclidean distance to highlight the transcriptome variance between the CD45RAintROintTSCM and CD45RA+TSCM subsets. One dimension explains 27% of the transcriptome variance between the two TSCM subsets.CD45RAintROintTSCM is shown in red, and CD45RA+TSCM is shown in green (n=7). F, Heatmap of pathways identified by Gene Set Enrichment Analysis (GSEA) that are significantly enriched in CD45RAintROintTSCM compared to CD45RA+TSCM, focusing on WNT signaling (Reactome). Agglutinated gene sets using predefined pathway tips revealed significant enrichment of these genes in the CD45RAintROintTSCM subset. The scale represents the NES score, and the red and blue squares indicate positive and negative enrichment, respectively. The columns represent the CD45RAintROintTSCM and CD45RA+TSCM subsets. G, Co-expression network highlighting significantly enriched tip-edge genes within the CD45RAintROintTSCM subset. Network connectivity and co-expression were inferred using the GeneMania algorithm. [Figure 5A-H]Plots showing the correlation between viral load control and ATI with CCR5 gene-edited TSCMs prior to ATI are shown. A. Plots showing viral load (VL) values ​​at week 22 (corresponding to week 16 of ATI) and historical pre-ART viral setpoint values ​​obtained from participant charts (data available for 14 out of 15 participants from study cohorts 1-5). Participants with extended ATI are shown in red. Wilcoxon rank-sum test p-values ​​are shown. B-C. Spearman rank correlation between changes in VL from week 22 to historical pre-ART viral setpoints and changes in CD4+ T cell count during peak expansion (weeks 1-3 post-infusion) (B) and the frequency of the "pentamer replication" marker per 106 PBMCs pre-ATI (week 6) (C). Participants with extended ATI are shown in red. Dashed lines represent 95% confidence intervals. Immunological and virological assays shown in panels d–h were performed on participants from cohorts 3–5, for whom cryopreserved cells were available for analysis. d–e are box plots with jitter overlaid showing the frequency of CCR5 gene-edited alleles determined by DNA sequencing for CD4+ T cell subsets (naive, CD45RA+TSCM, CD45RAintROintTSCM, TCM, TTM, and TEM) at 6 weeks post-infusion (pre-ATI) (D) and 22 weeks post-infusion (end of ATI) (E). The boxes show the median, first and third quartiles, and the whiskers extend to the maximum and minimum values. Participants with extended ATI are shown in red. n=7; +P<0.05; Wilcoxon rank-sum test. F-G: Spearman rank correlation between changes in VL (velocity level) from week 22 to the past pre-ART viral setting point and (f) CD45RAintROintTSCM and (G) CD45RA+TSCM before ATI (week 6). Participants who extended ATI are shown in red. Dashed lines represent 95% confidence intervals. (n=8; data on past pre-ART VL setting points was missing for participants 01-060 who were not included in the VL association analysis because they extended ATI).Three-dimensional scatter plots showing the change in VL as a function of H, CD45RAintROintTSCM cell number (w22 to past setpoint) and the frequency of CD8+ TTM cells producing IL-2 after gag peptide pool stimulation (using the time point with the maximum response, Tmax, for each participant) (n=8). Multivariate linear regression models were constructed using the CD45RAintROintTSCM number at week 6, as well as the frequency of CD8+ T cell subsets producing IFN-γ, TNF-α, and IL-2 cytokines after gag peptide pool stimulation following injection. The multivariate models that best predicted VL change included the CD45RAintROintTSCM number at week 6 (P=0.05) and HIV-specific CD8+ TTM cells producing IL-2 at Tmax (P=0.02). [Figure 6A-I]A plot shows that the level of CCR5 gene-edited TEM in ATI correlates with viral load control and reduces re-seeding in the TEMHIV reservoir. A, Box plot (n=7) showing the percentage of ZFN-induced CCR5 mutations unique to CD45RAintCD45ROintCCR7+CD27+(TSCM phenotype) in the SB-728-T product, detected in CD4+ T cell subsets at 6 and 22 weeks post-infusion. The box shows the median, first and third quartiles, and the whiskers extend to a distance of 1.5*IQR. Outliers are indicated by dots. B, Schematic diagram showing the dynamics of CCR5 gene-edited CD4+ T cell dynamics (see Materials and Methods for complete model details and assumptions). Model parameters are obtained by taking the geometric mean of five individual fitted outcomes for five participants with extended ATI duration. The parameters listed in the box represent parameters that showed a significant correlation (≧±0.5) with the cell population size obtained from sensitivity analysis tests performed in MATLAB® using 100,000 vials. C-E, Spearman rank correlation between the number of CCR5 gene-edited TEM cells at the end of ATI (week 22) and the number of CCR5 gene-edited CD45RA+TSCM (C), CD45RAintROintTSCM (D), and TCM (E) cells before ATI (week 6). Participants who extended ATI are shown in red. n=7. F, BL, box plots with jitter overlaid representing the frequency of integrated HIV DNA in TEM cells at weeks 6 and 22 after injection (n=7). The box shows the median, first and third quartiles, and the whiskers extend to the maximum and minimum values. Dots and lines are shown for all participants. Participants who extended ATI are shown in red. Wilcoxon rank-sum test p-values ​​are shown. Spearman rank correlation between the frequency of CCR5 gene-edited alleles in TEM during viremia (week 22) and changes in viral load from week 22 (16 weeks after ATI) to the previous pre-ART viral setpoint (G) and changes in the frequency of intermediate HIV DNA-carrying TEM cells from week 6 to week 22 (H). Participants with extended ATI are shown in red. Dashed lines represent 95% confidence intervals.n=6; For participants 01-060, who were not included in the VL-related analysis because their ATI was extended, data on past pre-ART VL setting points was missing. I. Spearman's rank correlation between the change in the frequency of TEM cells carrying intermediate HIV DNA from weeks 6 to 22 and the viral load level at week 22 (16 weeks after ATI). Participants with extended ATI are shown in red. The dashed line represents the 95% confidence interval. n=8. [Figure 7] This plot shows the size of the HIV reservoir in SB-728-0902 study participants at baseline. Correlation between the number of CD4+ T cells at baseline (BL) and the level of integrated HIV DNA at BL, as measured in purified CD4+ T cells. Spearman's rho(ρ) test was used. The dashed line represents the 95% confidence interval. [Figure 8A-E]This shows that a single infusion of SB-728-T led to a sustained increase in total CD4+ T cell count, an improvement in the CD4:CD8 ratio, and long-term persistence of CCR5 gene-edited cells. A shows CD4+ T cell counts at baseline (BL, 7 days before infusion), day 14, 3, 6, and 12 months, as well as at long-term follow-up points including the final follow-up at year 2 and 3-4 years. The mean is shown by the black line. Wilcoxon signed-rank test *P<0.05, **P<0.01. For panels A-C, participants in Cohort 1 (approximately 1E10 injected cells) are shown by blue symbols, Cohort 2 (approximately 2E10 injected cells) by green symbols, and Cohort 3 (approximately 3E10 injected cells) by red symbols. B shows the CD4:CD8 ratio at BL, day 14, 3, 6, 12 months, 2, and 3-4 years. The mean is shown by the black line. *P<0.05, **P<0.01; Wilcoxon signed-rank test. C. Post-injection pentamer replication and magnification of marked CD4+ T cells were estimated for all nine study participants during follow-up, as described in Materials and Methods. Gray areas represent data points where the magnification change is less than 1. D. Box plot with jitter overlaid for pentamer replication (marker of gene-edited cells) per 106 mononuclear cells from post-injection rectal biopsy. The box plot shows the 75th percentile (top), median (solid line in the box), and 25th percentile (bottom). Whiskers are drawn from minimum to maximum. E. Plot of pentamer replication marker per 106 PBMCs (black circles) and mononuclear cells (LNMC, squares) from lymph node biopsy after injection for three individuals in whom the pentamer replication marker was quantified in LNMCs. [Figure 9A-C]Plots illustrating the characteristics of the SB-728-T product are shown. A, levels of integrated HIV DNA in purified CD4+ T cells from pre-production leukocyte apheresis samples (BL) and post-production (SB-728-T product). The p-values ​​of the Wilcoxon signed-rank test are shown. Live CD3+CD4+ cells were gated on CD45RA and CD45RO, followed by CCR7 and CD27 to identify naive (CD45RA+CD45RO-CCR7+CD27+), CD45RAintCD45ROintTSCM-like cells, TCM (CD45RA-CD45RO+CCR7+CD27+), TTM (CD45RA-CD45RO+CCR7-CD27+), TEM (CD45RA-CD45RO+CCR7-CD27-), and CD45RA-CD45RO+CCR7+CD27- subsets. B. Frequency of CD4+ T cell subsets observed in the SB-728-T product. Lines represent the mean and standard deviation. C. Frequency of CCR5 gene-edited alleles in CD4+ T cell subsets of the SB-728-T product, measured by DNA sequencing of diverse CCR5 ZFN-induced mutations. Lines represent the mean and standard deviation. *P<0.05, **P<0.01; Wilcoxon signed-rank test. [Figure 10A-F]The plot shows that the frequency of CD58+CD95+ cells in the CD45RAintCD45ROint and CD45RA+CD45RO- subsets increased after injection, contributing to the persistence of CCR5 gene-edited cells. Histograms show the mean frequencies of cells expressing CD58 and CD95 in the CD45RAintCD45ROintCCR7+CD27+CD127+CD28+(CD45RAintROintTSCM, A) and CD45RA+CD45RO-CCR7+CD27+CD127+CD28+(CD45RA+TSCM, B) subsets at A-B, BL (n=6), mid-term (6-8 months, n=5), late-term (9-11 months, n=5), and long-term (3-4 years, n=6) post-injection time points. Error bars represent standard deviation. *P<0.05, **P<0.01; Mann-Whitney test. C-D. The frequency of CD58+CD95+ cells within the CD45RA+RO- and CD45RAintROint subsets at 3-4 years correlated with the estimated divergence of CCR5 gene-edited CD4+ T cells in PBMCs at long-term time (number of CCR5 gene-edited alleles at 3-4 years relative to the number (dose) of injected CCR5 gene-edited alleles). E. Longitudinal analysis of the number of CD45RA+RO-CCR7+CD27+CD127+CD28+CD58+CD95+ (referred to as CD45RA+RO-TSCM) and CD45RA+RO-CCR7+CD27+CD127+CD28+CD58-CD95- (referred to as naive) cells at BL (up to 3 months prior to injection), as well as at mid-term (6 months), late-term (8-11 months), and long-term time (3-4 years) post-injection for six subjects who underwent BL analysis. Longitudinal analysis of the number of CD45RAintROintCCR7+CD27+CD127+CD28+CD58+CD95+ (referred to as CD45RAintROintTSCM) and CD45RAintROintCCR7+CD27+CD127+CD28+CD58-CD95- (referred to as CD45RAintROintCD95-) cells in six subjects who underwent F and BL analysis, at BL (up to 3 months before injection), and at mid-term (6 months), late-term (8-11 months), and long-term (up to 44 months) post-injection time points. [Figure 11]This plot shows that CD45RAintROintTSCM cells have higher levels of CCR5 gene-editing alleles compared to other memory subsets. A box plot overlaid with jitter of CCR5 gene-editing allele frequencies in selected CD4+ T cell subsets 2–4 years post-injection, measured by DNA sequencing of diverse CCR5 ZFN-induced mutations. Lines represent the mean and standard deviation. The box plot shows the 75th percentile (top), median (solid line in the box), and 25th percentile (bottom). Whiskers are drawn from minimum to maximum. Wilcoxon signed-rank test p-values ​​are shown. [Figure 12A-B] This plot shows that CCR5 gene-edited T cells contribute to polyclonal and unbiased rearrangement of T cells. A. Bar graph showing TCR diversity in pre-production samples (CD4+ T cells purified from BL leukocyte apheresis samples, n=4), SB-728-T product (n=4), and purified CD4+ T cells at 7 days post-infusion (peak of CCR5 gene-edited cell proliferation) (n=3) and 7–9 months (n=4). Shannon entropy index was used to measure TCR clone diversity. NA = samples were not available at this point. B. CCR5 gene-edited allele diversity in CD4+ T cell subsets from SB-728-T product compared to subsets from long-term post-infusion time points (3–4 years) using Shannon entropy index (n=8). Lines represent the mean and standard deviation. Wilcoxon signed-rank test p-values ​​are shown. [Figure 13A-B]The graph shows that CD45RAintROintTSCM cells are a small contributor to the total HIV reservoir. Selected CD45RA-CD45RO+ subsets in the SB-728-T product (TCM, n=9; TTM, n=8; TEM, n=9; and CCR7+CD27, n=9), as well as the CD45RAintCD45ROint subset (total CD45RAintCD45ROint, n=9; and CD45RAintCD45ROintCCR7+CD27+, n=9) (A), and selected CD45RAintCD45ROintCCR7+CD27+ cells 3-4 years after infusion. Histogram showing the mean levels of integrated HIV DNA (log10 copies / 10⁶ cells) in the 45RA-CD45RO+ subset ((TCM, n=8; TTM, n=7; and TEM, n=8), the CD45RAintCD45ROint subset (CD95+; CD45RAintROintTSCM, n=8; and CD95-, n=8), and the CD45RA+CD45RO- subset (CD95+; CD45RA+TSCM), n=6; and CD95-; naive, n=7) (B). Error bars represent standard deviation. *P<0.05, **P<0.01; Wilcoxon signed-rank test. [Figure 14A-B] The following plots demonstrate that CD45RAintROintTSCM cells constitute a distinct population from the aforementioned CD45RA+TSCM subset. A. Multidimensional scaling (MDS plot) is used to highlight the transcriptome variance of CD4+ T cell subsets 3-4 years after injection. Euclidean distance is used, and dimensionality reduction for top mutant genes based on ANOVA (analysis of variance, F-test, n=3358 transcripts, P≦0.05) is represented. One dimension explains 50% of the variance between CD4+ T cell subsets. Different subsets are represented by different symbols. n=7 samples per cell subset. B. Bar graph representing the number of genes differentially expressed between CD45RAintROintTSCM and CD45RA+TSCM, TCM, or TEM (DEG, P<0.05). [Figure 15A-B]This plot shows the viral load of subjects who underwent analytical treatment interruption (ATI) after SB-728-T infusion in the 1101 study. A. Summary of the 1101 clinical trial. Subjects received escalating dose cytoxane (CTX) pre-adjustment two days prior to infusion. Subjects received ATI at 16-week intervals starting 6 weeks after infusion. B. Viral load (VL) is shown for 9 subjects who resumed ART by week 22, and 6 subjects whose ATI was extended (VL remained below 10,000 copies / mL and CD4+ T cell count exceeded 500 cells / μl). The red line represents the resumption of ART. [Figure 16A-B] Plots showing the distribution and cell count of CD4+ T cell subsets post-infusion (week 6) and post-treatment discontinuation (week 22) in the 1101 study are shown. The frequency (A) and cell count (B) of CD4 T cell subsets (naive, CD45RA+TSCM, CD45RAintROintTSCM, TCM, TTM, and TEM) at 6 weeks post-infusion (pre-ATI) and 22 weeks post-infusion (end of ATI) are shown (n=9). The p-values ​​of the Wilcoxon signed-rank test are shown. [Figure 17] This shows the frequency of CCR5 gene-edited cells in CD4+ T cell subsets after ATI in the 1101 study. The frequency of CCR5 gene-edited alleles, determined by DNA sequencing, is shown for CD4+ T cell subsets (CD45RA+ TSCM, CD45RAintROintTSCM, TCM, and TEM) at 6 weeks post-infusion (pre-ATI), 22 weeks, 7 / 8 months, and 12 months (during ATI) for participants whose ATI was extended to at least 12 months. [Modes for carrying out the invention]

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention pertains.

[0017] As used herein, each of the following terms has the meaning associated with it in this section.

[0018] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "one element" means one or more elements.

[0019] As used herein, "about" when referring to measurable values ​​such as quantity or temporal duration means that it includes variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, and such variations are appropriate for carrying out the disclosed method.

[0020] As used herein, “activation” refers to the state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with inducible cytokine production and detectable effector function. The term “activated T cell” refers, among other things, to a T cell undergoing cell division.

[0021] As used herein, the term “antibody” refers to an immunoglobulin molecule capable of specifically binding to a particular epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, or they can be the immunoactive moiety of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies of the present invention may exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies (Harlow et al., 1988, Houston et al., 1988, Bird et al., 1988).

[0022] As used herein, the terms “antigen” or “Ag” are defined as molecules that trigger an immune response. This immune response may involve either antibody production or activation of specific immune-qualified cells, or both. A skilled person will understand that virtually any macromolecule, including proteins or peptides, can serve as an antigen. Furthermore, antigens can be recombinant or derived from genomic DNA. A skilled person will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that triggers an immune response, therefore, encodes an “antigen” as the term is used herein. Furthermore, a person skilled in the art will understand that antigens do not need to be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences can be arranged in various combinations to trigger a desired immune response. Furthermore, a skilled person will understand that antigens do not need to be encoded by a “gene” at all. It is readily apparent that antigens can be generated and synthesized, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0023] As used herein, the term “homemade” means any material derived from the same individual that is later reintroduced into that individual.

[0024] "Homogenous grafts" refer to grafts derived from different animals of the same species.

[0025] "Heterogeneous" refers to grafts derived from animals of different species.

[0026] As used herein, “effective dose” means the amount that provides a therapeutic or preventive benefit.

[0027] As used herein, the term “expression” is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0028] As used herein, the term “specifically binds” means a molecule, such as an antibody, that recognizes and binds to another molecule or feature, but does not substantially recognize or bind to any other molecule or feature in the sample.

[0029] As used herein, the term “inhibit” means to reduce by a measurable amount, or completely prevent, the expression, stability, function, or activity of a molecule, reaction, interaction, gene, mRNA, and / or protein. Inhibitors are compounds, e.g., antagonists, that partially or completely block, reduce, prevent, delay, inactivate, desensitize, or downmodulate a stimulus that binds to the stability, expression, function, and activity of a protein, gene, and mRNA.

[0030] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably and refer to deoxyribonucleotides or ribonucleotide polymers in linear or circular conformations, in either single-stranded or double-stranded form. For the purposes of this disclosure, these terms should not be construed as limiting with respect to the length of the polymer. These terms may encompass known analogues of natural nucleotides, as well as nucleotides modified with bases, sugars, and / or phosphate moieties (e.g., phosphorothioate skeletons). Generally, analogues of a particular nucleotide have the same base-pairing specificity; i.e., an analogue of A will base-pair with T.

[0031] The terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to polymers of amino acid residues. This term also applies to amino acid polymers, where one or more amino acids are chemical analogues or modified derivatives of corresponding naturally occurring amino acids.

[0032] "Binding" refers to sequence-specific non-covalent interactions between macromolecules (e.g., between a protein and a nucleic acid, or between two nucleic acids). Not all components of the binding interaction need to be sequence-specific (e.g., contact with phosphate residues in the DNA backbone), as long as the interaction as a whole is sequence-specific. Such interactions are generally 10 -6 M -1 The following dissociation constant (K d ) is characterized by. "Affinity" refers to the strength of the bond, and an increase in binding affinity is associated with a lower K d It is correlated with this.

[0033] The terms “chimeric RNA,” “chimeric guide RNA,” “guide RNA,” “single guide RNA,” and “synthetic guide RNA” are used interchangeably and refer to polynucleotide sequences including guide sequences, tracr sequences, and tracr mate sequences. The term “guide sequence” refers to a sequence of approximately 10–30 (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) base pairs within the guide RNA that identifies the target site, and can be used interchangeably with the terms “guide” or “spacer.” The term “tracr mate sequence” can also be used interchangeably with the term “direct repeat.”

[0034] "Complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, either through conventional Watson-Crick or other unconventional methods. The complementarity percentage indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (for example, 5, 6, 7, 8, 9, and 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Perfectly complementary" means that all adjacent residues in one nucleic acid sequence can form hydrogen bonds with the same number of adjacent residues in the second nucleic acid sequence. As used herein, “substantially complementary” refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% for regions of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or two nucleic acids that hybridize under stringent conditions.

[0035] A "binding protein" is a protein that can bind to another molecule. Binding proteins can, for example, bind to DNA molecules (DNA-binding proteins), RNA molecules (RNA-binding proteins), and / or protein molecules (protein-binding proteins). Protein-binding proteins can bind to themselves (forming homodimers, homotrimers, etc.) and / or to one or more molecules of one or more different proteins. Binding proteins can possess multiple types of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding, and protein-binding activity.

[0036] A "zinc finger DNA-binding protein" (or binding domain) is a domain within a larger protein that binds sequence-specifically to DNA via a protein or one or more zinc fingers. These are regions of amino acid sequences within a binding domain whose structure is stabilized by the coordination of zinc ions. The term zinc finger DNA-binding protein is often abbreviated as zinc finger protein or ZFP.

[0037] A "TALE DNA-binding domain" or "TALE" is a polypeptide containing one or more TALE repeat domains / units. The repeat domains are involved in the binding of the TALE to its congenerate target DNA sequences. A single "repeat unit" (also called a "repeat") is typically 33-35 amino acids long and exhibits at least some degree of sequence homology to other TALE repeat sequences within naturally occurring TALE proteins.

[0038] Zinc finger and TALE binding domains can be "engineered" to bind to a given nucleotide sequence, for example, by manipulating the recognition helix region of a naturally occurring zinc finger or TALE protein (modifying one or more amino acids). Therefore, an engineered DNA-binding protein (zinc finger or TALE) is a protein that does not exist in nature. Non-limiting examples of methods for manipulating DNA-binding proteins are design and selection. An engineered DNA-binding protein is a protein that does not exist in nature, and its design / composition is based primarily on reasonable criteria. Reasonable criteria for design include the application of substitution rules and computerized algorithms to process information in databases storing existing ZFP and / or TALE designs as well as binding data. See, for example, U.S. Patents 8,586,526, 6,140,081, 6,453,242, and 6,534,261. See also WO98 / 53058, WO98 / 53059, WO98 / 53060, WO02 / 016536 and WO03 / 016496.

[0039] "Selected" zinc finger proteins, or TALEs, are proteins not found in nature, and their production arises primarily from empirical processes such as phage display, interaction trapping, or hybrid selection. See, for example, U.S. Patents 8,586,526, 5,789,538, 5,925,523, 6,007,988, 6,013,453, 6,200,759, as well as WO95 / 19431, WO96 / 06166, WO98 / 53057, WO98 / 54311, WO00 / 27878, WO01 / 60970, WO01 / 88197, and WO02 / 099084.

[0040] "Recombination" refers to the process of exchanging genetic information between two polynucleotides. For the purposes of this disclosure, "homologous recombination (HR)" refers to a specific form of such exchange that occurs, for example, during the repair of double-strand breaks in cells via homology-directed repair mechanisms. This process requires homology of nucleotide sequences and uses a "donor" molecule to template the repair of a "target" molecule (i.e., the molecule that has experienced a double-strand break), leading to the transfer of genetic information from the donor to the target, and is therefore known in various ways as "non-crossover gene conversion" or "short tract gene conversion." While we do not wish to be bound by any particular theory, such transfers may involve "synthesis-dependent strand annealing," which is used to correct the mismatch of heteroduplex DNA formed between the broken target and the donor, and / or to resynthesize the genetic information and / or related processes that the donor will become part of the target. Such specific HRs often result in alterations of the target molecule's sequence, such that some or all of the donor polynucleotide sequence is incorporated into the target polynucleotide.

[0041] In the methods of this disclosure, one or more target nucleases described herein (e.g., CRISPR / Cas) can create a double-strand break in a target sequence (e.g., cellular chromatin) at a given site, and introduce a “donor” polynucleotide having homology to the nucleotide sequence of the break region into the cell. The presence of the double-strand break has been shown to facilitate integration of the donor sequence. The donor sequence may be physically incorporated, or the donor polynucleotide may be used as a template for repairing the break via homologous recombination, with all or part of the nucleotide sequence in the donor being introduced into the cellular chromatin. Thus, the first sequence of cellular chromatin can be modified and, in certain embodiments, converted to a sequence present in the donor polynucleotide. Therefore, the use of the terms “substitute” or “replace” can be understood to refer to the substitution of one nucleotide sequence with another nucleotide sequence (i.e., sequence substitution in an informational sense) and does not necessarily require physical or chemical substitution from one polynucleotide to another.

[0042] In any of the methods described herein, additional pairs of CRISPR / Cas nucleases and / or zinc finger or TALEN proteins may be used to perform additional double-strand breaks at additional target sites within the cell.

[0043] In a particular embodiment of the method for targeted recombination and / or substitution and / or modification of sequences in a region of interest of cellular chromatin, a chromosomal sequence is modified by homologous recombination with an exogenous "donor" nucleotide sequence. Such homologous recombination is stimulated by the presence of a double-strand break in cellular chromatin if a homologous sequence is present in the region of the break.

[0044] In any of the methods described herein, the exogenous nucleotide sequence ("donor sequence" or "transgene") may contain sequences that are homologous but not identical to the genomic sequence in the region of interest, thereby stimulating homologous recombination to insert the non-identical sequence in the region of interest. Thus, in certain embodiments, the portion of the donor sequence homologous to the sequence in the region of interest exhibits approximately 80–99% (or any integer between those values) sequence identity with respect to the genomic sequence being replaced. In other embodiments, for example, if the donor sequence of more than 100 consecutive base pairs differs from the genomic sequence by only one nucleotide, the homology between the donor and the genomic sequence is higher than 99%. In certain cases, the non-homologous portion of the donor sequence may contain sequences not present in the region of interest, thereby introducing a new sequence into the region of interest. In these examples, the non-homologous sequence is generally adjacent to a sequence of 50–1,000 base pairs (or any integer between those values) or any number of base pairs greater than 1,000 that is homologous or identical to the sequence in the region of interest. In another embodiment, the donor sequence is non-homologous to the first sequence and is inserted into the genome by a non-homologous recombination mechanism.

[0045] Any of the methods described herein can be used to partially or completely inactivate one or more target sequences within a cell by targeted integration of a donor sequence that disrupts the expression of the gene of interest. Cell lines having partially or completely inactivated genes are also provided.

[0046] Furthermore, one or more exogenous sequences can be integrated using the targeted integration methods described herein. Exogenous nucleic acid sequences may include, for example, one or more genes or cDNA molecules, or any type of coding or non-coding sequence, as well as one or more regulatory elements (e.g., promoters). In addition, exogenous nucleic acid sequences can produce one or more RNA molecules (e.g., small hairpin RNA (shRNA), inhibitory RNA (RNAis), microRNA (miRNA), etc.).

[0047] "Cutting" refers to the cleavage of the covalent backbone of a DNA molecule. Cutting can be initiated by a variety of methods, including but not limited to enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand breaks are possible, and a double-strand break can occur as a result of two different single-strand break events. DNA breaks can result in the production of either blunt or adherent ends. In certain embodiments, fusion polypeptides are used for targeted double-strand DNA breaks.

[0048] A “cleavage half-domain” is a polypeptide sequence that, together with a second polypeptide (identical or different), forms a complex having cleavage activity (preferably double-strand cleavage activity). The terms “first and second cleavage half-domains,” “+ and - cleavage half-domains,” and “right and left cleavage half-domains” are used interchangeably to refer to pairs of dimerizing cleavage half-domains.

[0049] A “manipulated cleavage half-domain” is a cleavage half-domain modified to form an obligatory heterodimer with another cleavage half-domain (e.g., another manipulated cleavage half-domain). See also U.S. Patent Publications 2005 / 0064474, 2007 / 0218528, 2008 / 0131962, and 2011 / 0201055 (all incorporated herein by reference).

[0050] The term “sequence” refers to a nucleotide sequence of any length, which may be DNA or RNA, may be linear, circular, or branched, and may be single-stranded or double-stranded. The term “donor sequence” refers to a nucleotide sequence inserted into the genome. Donor sequences may be of any length, for example, 2 to 10,000 nucleotides (or any integer between or greater than these), preferably about 100 to 1,000 nucleotides (or any integer between these), and more preferably about 200 to 500 nucleotides.

[0051] A “homologous non-identical sequence” refers to a first sequence that shares some degree of sequence identity with a second sequence, but whose sequence is not identical to the sequence identity of the second sequence. For example, a polynucleotide containing the wild-type sequence of a mutant gene is homologous and non-identical to the sequence of the mutant gene. In certain embodiments, the degree of homology between the two sequences is sufficient to enable homologous recombination between them using normal cellular mechanisms. The two homologous non-identical sequences can be of any length, and their degree of non-homonymy can be as small as a single nucleotide (e.g., for correcting genomic point mutations by targeted homologous recombination) or as large as 10 kilobases or more (e.g., for inserting a gene into a predetermined ectopic site on a chromosome). The two polynucleotides containing homologous non-identical sequences do not need to be the same length. For example, 20 to 10,000 nucleotides or nucleotide pairs of exogenous polynucleotides (i.e., donor polynucleotides) can be used.

[0052] Techniques for determining the identity of nucleic acid and amino acid sequences are known in the art. Typically, such techniques involve determining the nucleotide sequence of a gene's mRNA and / or the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this manner. Generally, identity refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Two or more sequences (polynucleotides or amino acids) can be compared by determining their identity percentage. Whether nucleic acid sequences or amino acid sequences, the identity percentage of two sequences is the number of perfect matches between the two aligned sequences divided by the length of the shorter sequence and multiplied by 100. Approximate alignment of nucleic acid sequences is provided by the local homology algorithm in Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm is applied to amino acid sequences by using a scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, DC, USA, and can be normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm for determining the identity percentage of sequences is provided by Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application. Suitable programs for calculating the identity or similarity percentage between sequences are commonly known in the art; for example, another alignment program used with default parameters is BLAST.For example, BLASTN and BLASTP can be used with the following default parameters: gene code=standard, filter=none, strand=both, cutoff=60, expect=10, matrix=BLOSUM62, description=50 sequences, sort=HIGH SCORE, database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swiss protein+Spupdate+PIR. Details of these programs can be found on the internet. With respect to the sequences described herein, the desired degree of sequence identity ranges from approximately 80% to 100% and any integer value in between. Typically, the percentage of identity between sequences is at least 70-75%, preferably 80-82%, more preferably 85-90%, even more preferably 92%, even more preferably 95%, and most preferably 98% sequence identity.

[0053] Alternatively, the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that allow for the formation of stable double helixes between homologous regions, followed by digestion with a single-strand specific nuclease, and sizing of the digested fragments. Two nucleic acids, or two polypeptide sequences, are substantially homologous to each other if, as determined using the methods described above, the sequences exhibit sequence identity of at least about 70%–75%, preferably 80%–82%, more preferably 85%–90%, even more preferably 92%, even more preferably 95%, and most preferably 98% over a defined length of the molecule. As used herein, substantially homologous also refers to sequences that exhibit complete identity to a given DNA or polypeptide sequence. Substantially homologous DNA sequences can be identified, for example, in Southern hybridization experiments under stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is known to those skilled in the art. For example, see Sambrook et al., above; Nucleic Acid Hybridization: A Practical Approach, editors B. D. Ames and S. J. Higgins, (1985) Oxford; Washington, DC; IRL Press).

[0054] Selective hybridization of two nucleic acid fragments can be determined as follows: The degree of sequence identity between the two nucleic acid molecules affects the efficiency and intensity of the hybridization event between such molecules. Partially identical nucleic acid sequences will at least partially inhibit the hybridization of completely identical sequences to the target molecule. Inhibition of hybridization of completely identical sequences can be evaluated using hybridization assays well known in the art (e.g., Southern (DNA) blot, Northern (RNA) blot, solution hybridization, etc.; see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989), Cold Spring Harbor, NY). Such assays can be performed using varying degrees of selectivity, for example, by using conditions that vary from low to high stringency. When low stringency conditions are used, the absence of nonspecific binding can be assessed using a secondary probe that lacks even partial sequence identity (e.g., a probe with less than approximately 30% sequence identity with the target molecule), thereby ensuring that if there are no nonspecific binding events, the secondary probe will not hybridize to the target.

[0055] Chromatin is the nuclear protein structure that makes up the cell genome. Cellular chromatin is composed of nucleic acids, mainly DNA, as well as proteins, including histone and non-histone chromosomal proteins. The majority of eukaryotic cellular chromatin exists in the form of nucleosomes, the nucleosome core containing approximately 150 base pairs of DNA associated with an octamer containing two each of histones H2A, H2B, H3, and H4, with linker DNA (of variable length depending on the organism) extending between the nucleosome cores. The histone H1 molecule is generally associated with the linker DNA. For the purposes of this disclosure, the term “chromatin” means to encompass all types of cellular nuclear proteins in both prokaryotes and eukaryotes. Cellular chromatin includes both chromosomal chromatin and episomal chromatin.

[0056] A "chromosome" is a chromatin complex that contains all or part of a cell's genome. A cell's genome is often characterized by a karyotype, which is the set of all the chromosomes that make up the cell's genome. A cell's genome may contain one or more chromosomes.

[0057] An "episome" is a structure containing nucleic acids that replicate, a nuclear protein complex, or other nucleic acids that are not part of a cell's chromosomal karyotype. Examples of episomes include plasmids and certain viral genomes.

[0058] An "accessible region" is a region within cellular chromatin where a target site present in a nucleic acid can be bound by an exogenous molecule that recognizes the target site. While we do not wish to be constrained by any particular theory, accessible regions are generally considered to be regions not packaged within the nucleosome structure. The distinct structure of accessible regions can often be detected by chemical and enzymatic probes, such as sensitivity to nucleases.

[0059] A "target site" or "target sequence" is a nucleic acid sequence that defines the portion of the nucleic acid to which a binding molecule will bind, provided that sufficient conditions for binding are present.

[0060] "Exogenous" molecules are those that are not normally present in cells but can be introduced into cells by one or more genetic, biochemical, or other means. "Normal presence within a cell" is determined with respect to specific developmental stages and environmental conditions of the cell. For example, a molecule present only during muscle embryonic development is an exogenous molecule with respect to adult muscle cells. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to cells that have not experienced heat shock. Exogenous molecules may include, for example, a functioning version of a dysfunctional endogenous molecule, or a dysfunctional version of a normally functioning endogenous molecule.

[0061] Exogenous molecules may include, among other things, small molecules produced by combinatorial chemical processes, or macromolecules such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, any modified derivatives of the above molecules, or any complexes containing one or more of the above molecules. Nucleic acids include DNA and RNA, and may be single-stranded or double-stranded, linear, branched, or circular, and may be of any length. Nucleic acids include those that can form double helixes, and similarly, those that form triple helixes. See, for example, U.S. Patents 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA-binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyralases, and helicases. Therefore, this term includes "transgenes" or "genes of interest," which are exogenous sequences introduced into cells.

[0062] Exogenous molecules can be molecules of the same type as endogenous molecules, such as exogenous proteins or nucleic acids. For example, exogenous nucleic acids may include infectious viral genomes, plasmids or episomes introduced into cells, or chromosomes that are not normally present in cells. Methods for introducing exogenous molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate coprecipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer. Exogenous molecules can also be molecules of the same type as endogenous molecules, but may originate from a different species than the cell from which it originates. For example, human nucleic acid sequences can be introduced into cell lines originally derived from mice or hamsters. Methods for introducing exogenous molecules into plant cells are known to those skilled in the art and include, but are not limited to, protoplast transformation, silicon carbide (e.g., WHISKERS®), Agrobacterium-mediated transformation, lipid-mediated transfer (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment (e.g., using a “gene gun”), calcium phosphate coprecipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer.

[0063] In contrast, "endogenous" molecules are molecules that are normally present in specific cells at specific developmental stages under specific environmental conditions. For example, endogenous nucleic acids may include chromosomes, mitochondrial genomes, chloroplasts or other organelles, or naturally occurring episomal nucleic acids. Additional endogenous molecules may include proteins, such as transcription factors and enzymes.

[0064] As used herein, the term “exogenous nucleic acid products” includes both polynucleotide and polypeptide products, e.g., transcripts (polynucleotides such as RNA) and translation products (polypeptides).

[0065] A “fusion” molecule is a molecule in which two or more subunit molecules are linked, preferably by covalent bonds. The subunit molecules may be molecules of the same chemical type or molecules of different chemical types. Examples of the first type of fusion molecule include, but are not limited to, fusion proteins (e.g., fusions between a ZFP or TALE DNA-binding domain and one or more activation domains) and fusion nucleic acids (e.g., nucleic acids encoding the above-mentioned fusion proteins). Examples of the second type of fusion molecule include, but are not limited to, fusions between triple-helical nucleic acids and polypeptides, and fusions between a sub-groove binder and nucleic acids. A “fusion polypeptide” is a polypeptide comprising a polypeptide or a portion thereof (e.g., one or more domains) fused to or bound to a heterologous polypeptide. Examples of fusion polypeptides include immunoadhesins that combine a portion of the Cas protein with an immunoglobulin sequence, and epitope-tagged polypeptides that may contain the Cas protein, e.g., or a portion thereof fused to a “tagged polypeptide.” A tagged polypeptide has enough residues to provide an epitope from which an antibody can be produced, but is short enough not to interfere with the nuclease activity of Cas. A suitable tag polypeptide generally has at least 6 amino acid residues, and usually about 6 to 60 amino acid residues.

[0066] The expression of fusion proteins in cells may result from the delivery of the fusion protein to the cell, or from the delivery of the polynucleotide encoding the fusion protein to the cell, where the polynucleotide is transcribed, the transcript is translated, and the fusion protein is produced. Trans-splicing, polypeptide cleavage, and polypeptide ligation may also be involved in the expression of proteins in cells. Methods for the delivery of polynucleotides and polypeptides to cells are presented elsewhere in this disclosure.

[0067] For the purposes of this disclosure, “gene” includes the DNA region that codes for a gene product (see below), as well as all DNA regions that regulate the production of the gene product, whether such regulatory sequences are adjacent to the coding and / or transcription sequences. Thus, a gene includes, but is not limited to, the promoter sequence, terminator, translation regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus regulatory regions. “Engineered gene” means a gene that has been modified in any way to be non-identical to a wild-type gene. Modifications may take the form of targeted deletions, insertions, and truncations. An engineered gene may contain coding sequences from two heterologous genes or may contain a synthetic gene sequence. An engineered gene may also contain coding sequence changes that are silent in the protein sequence (e.g., codon optimization). An engineered gene may also contain a gene in which regulatory sequences have been modified.

[0068] "Gene expression" refers to the process of converting the information contained in a gene into a gene product. A gene product can be a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or the direct transcript of a protein produced by the translation of mRNA. Gene products also include RNA modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified by processes such as methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0069] "Regulation" of gene expression refers to a change in gene activity. Regulation of expression may include, but is not limited to, gene activation and gene repression. Expression can be regulated using genome editing (e.g., cutting, modification, inactivation, random mutation). Gene inactivation refers to a reduction in gene expression compared to cells without the CRISPR / Cas system described herein. Therefore, gene inactivation may be partial or complete.

[0070] A "region of interest" is any region of cellular chromatin, such as a gene or a non-coding sequence within or adjacent to a gene, where binding to an exogenous molecule is desirable. Binding may be for the purpose of targeted DNA cleavage and / or targeted recombination. Regions of interest may be located, for example, in chromosomes, episomes, organelle genomes (e.g., mitochondria, chloroplasts), or infectious viral genomes. Regions of interest may be within the coding region of a gene, within a transcribed non-coding region such as a leader sequence, trailer sequence, or intron, or within an untranscribed region either upstream or downstream of a coding region. Regions of interest can be as short as a single nucleotide pair or up to 2,000 nucleotide pairs, or any integer value of nucleotide pairs.

[0071] Eukaryotic cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells, and human cells (e.g., T cells).

[0072] The terms “functional linkage” and “functionally linked” (or “operably linked”) are used interchangeably with respect to the juxtaposition of two or more components (such as sequence elements) arranged to take into account the possibility that both components function correctly and that at least one of the components mediates the function exerted by at least one of the other components. For example, if a transcriptional regulatory sequence controls the level of transcription of a coding sequence in response to the presence or absence of one or more transcriptional regulators, then the transcriptional regulatory sequence, such as a promoter, is functionally linked to the coding sequence. Transcriptional regulatory sequences are generally functionally linked to a coding sequence in cis, but do not need to be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is functionally linked to a coding sequence even if it is not adjacent.

[0073] With respect to fusion polypeptides, the term "functionally linked" can refer to the fact that each component links with other components to perform the same function as if they were not linked. For example, in the case of a fusion polypeptide in which a Cas DNA-binding domain is fused to an activation domain, the Cas DNA-binding domain and the activation domain are functionally linked if, in the fusion polypeptide, the Cas DNA-binding domain portion can bind to its target site and / or its binding site, while the activation domain can upregulate gene expression. In the case of a fusion polypeptide in which a Cas DNA-binding domain is fused to a cleavage domain, the Cas DNA-binding domain and the cleavage domain are functionally linked if, in the fusion polypeptide, the Cas DNA-binding domain portion can bind to its target site and / or its binding site, while the cleavage domain can cleave DNA near the target site.

[0074] A “functional fragment” of a protein, polypeptide, or nucleic acid is a protein, polypeptide, or nucleic acid whose sequence is not identical to that of the full-length protein, polypeptide, or nucleic acid, but which retains the same function as the full-length protein, polypeptide, or nucleic acid. A functional fragment may have more, fewer, or the same number of residues as the corresponding native molecule and / or may contain one or more amino acid or nucleotide substitutions. Methods for determining the function of nucleic acids (e.g., coding function, ability to hybridize to another nucleic acid) are well known in the art. Similarly, methods for determining the function of proteins are well known. For example, the DNA-binding function of a polypeptide can be determined, for example, by filter binding, electrophoretic mobility shift, or immunoprecipitation assay. DNA cleavage can be analyzed by gel electrophoresis. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assay, or both genetic and biochemical complementarity. For example, see Fields et al. (1989) Nature 340:245-246, U.S. Patent No. 5,585,245, and PCT WO98 / 44350.

[0075] A "vector" is capable of transferring a gene sequence into target cells. Typically, "vector constructs," "expression vectors," and "gene transfer vectors" refer to any nucleic acid construct that can direct the expression of a gene of interest and transfer a gene sequence into target cells. Therefore, this term includes cloning and expression vehicles, as well as integration vectors.

[0076] A “reporter gene” or “reporter sequence” preferably refers to any sequence that produces a protein product that is readily measurable, although this is not necessarily the case in routine assays. Suitable reporter genes include, but are not limited to, sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding colored, fluorescent, or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins that mediate enhanced cell proliferation and / or gene amplification (e.g., dihydrofolate reductase). Epitope tags include, for example, FLAG, His, myc, Tap, HA, or one or more copies of any detectable amino acid sequence. An “expression tag” includes a sequence encoding a reporter that can be operably ligated to a desired gene sequence to monitor the expression of the gene of interest.

[0077] The terms “subject” and “patient” are used interchangeably and refer to human patients and mammals such as non-human primates, as well as experimental animals such as rabbits, dogs, cats, rats, mice, and other animals. Therefore, as used herein, the terms “subject” or “patient” mean any mammalian patient or subject to whom the stem cells of the present invention can be administered. Subjects of the present invention include, for example, subjects exposed to one or more chemical toxins, including neurotoxins.

[0078] As used herein, the term “therapeutic” means treatment and / or prevention. Therapeutic effects are obtained by suppression, remission, or eradication of the disease.

[0079] The term “therapeutic dose” refers to the amount of a compound of interest that would induce a biological or medical response in a tissue, system, or subject, as sought by researchers, veterinarians, physicians, or other clinicians. “Therapeutic dose” includes an amount of the compound sufficient to prevent, or to some extent alleviate, the onset of one or more signs or symptoms of the disorder or disease being treated, when administered. The therapeutic dose will vary depending on the compound being treated, the disease and its severity, as well as age, weight, etc. “To treat” a disease, as used herein, means to reduce the frequency or severity of at least one sign or symptom of the disease or disorder experienced by the subject.

[0080] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process by which an exogenous nucleic acid is transferred to or introduced into a host cell. A “transfected,” “transformed,” or “transduced” cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Cells include the primary target cell and its progeny.

[0081] A “vector” is a composition of substances containing isolated nucleic acids that can be used to deliver the isolated nucleic acids into the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes plasmids or viruses that autonomously replicate. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, and retroviral vectors.

[0082] Scope: Throughout this disclosure, various aspects of the invention can be presented in range form. It should be understood that descriptions in range form are merely for convenience and conciseness and should not be interpreted as inflexible limitations on the scope of the invention. Therefore, range descriptions should be considered to specifically disclose all possible subranges, not just the individual numbers within that range. For example, a range description such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and the individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0083] The embodiments described herein are CD45RA int CD45RO int phenotypic CD4 T cells and CD8 T cells (CD4 / CD8 T cells), CD45RA int CD45RO int This study concerns long-lived, enriched populations of genetically modified and / or altered CD4 / CD8 T cells with a specific phenotype, and their use in the treatment of latent HIV infection in HIV-infected subjects, particularly those receiving and / or continuing antiretroviral therapy. The subset of CD4 / CD8 T cells was found to possess the phenotypic and molecular attributes of long-lived, pluripotent stem cells. Similar to other known stem cell populations, this subset has a low metabolic profile (upregulation of fatty acid metabolism and oxidative phosphorylation, as well as downregulation of cellular circulation pathways), retains self-renewal capacity, and can differentiate into effector cells. This subset primarily exhibits intermediate co-expression of CD45RA and CD45RO (CD45RA int CD45RO int ) is a characteristic of CD45RA int CD45RO int CD4 / CD8 T cells exhibiting the phenotype can also express CD95(Fas), CD127(IL7R), and CD27. Addition of low doses of cytokines IL-7 and IL-15 can induce CD45RA int CD45RO intWhile this may lead to the formation of enriched populations of phenotypic CD4 / CD8 cells, high doses of cytokines IL-7 and IL-15 may lead to effector differentiation of cells.

[0084] In some embodiments, CD45RA int CD45RO int CD4 / CD8 T cells exhibiting the phenotype can be genetically modified to lack the functional CCR5 and / or CXCR4 HIV co-receptors. CD45RA in HIV-infected subjects. int CD45RO int Administration of autologous CD4 / CD8 T cells with phenotypic CCR5 and / or CXCR4 gene editing may result in a sustained increase in CD4+ T cell count, restoration of T cell homeostasis, and a significant reduction in HIV reservoir size in subjects.

[0085] When transferring or administering to the target, CD45RA int CD45RO int Phenotypic enriched populations of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells possess the ability to persist or survive for extended periods in the target population. Persistence may correlate with the effectiveness of therapeutic T cell transplantation in the treatment of diseases such as HIV infection. Higher persistence of therapeutic T cells increases the likelihood of a more effective treatment regimen. Therefore, CD45RA int CD45RO int Long-lived, self-renewing, and pluripotent CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells possessing a specific phenotype can reduce production costs, promote effector differentiation, and increase the therapeutic efficiency of latent HIV infection in the target population. Furthermore, the frequency of these cells in currently available HIV therapeutic products can be used as a biomarker to predict the success of interventions.

[0086] In some embodiments, CD45RA int CD45RO intEnriched populations of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells exhibiting the phenotype were CD45RA after administration to the target. int CD45RO int CD45RA can persist in vivo for at least 1, 2, 3, 4, 5, 6, 12, 24, 36, 48, or 72 months longer than T cells without the phenotype. int CD45RO int Phenotypic enriched populations of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells may also have increased engraftment ability in subjects after administration. Specifically, CD45RA int CD45RO int Phenotypic enriched populations of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells may have an increased ability to engraft in unadapted recipients.

[0087] The term "engraftment" refers to the ability of transplanted cells to establish themselves in the recipient and survive immediately after transplantation. Therefore, engraftment is assessed in the short period after transplantation. For example, engraftment may refer to the number of cells derived from transplanted cells that can be detected in the recipient at the earliest possible time in the initial in vivo evaluation of an experiment, clinical trial, or treatment protocol, such as transplanted cells or their offspring. In one embodiment, engraftment is assessed at 0–12, 0–24, 0–48, or 0–72 hours after transplantation. In another embodiment, engraftment is assessed at approximately 1, 2, 3, 4, 5, 6, 12, 24, 36, 48, 60, or 72 hours after transplantation. In a preferred embodiment, engraftment is assessed at approximately 12 hours after transplantation.

[0088] Figure 1 shows CD45RA, which can be genetically modified to lack the functional CCR5 and / or CXCR4 HIV co-receptors. int CD45RO intA flowchart showing a method for generating an enriched population of CD4 / CD8 T cells having a phenotype is shown. In this method, in step 10, a naive population of T cells is isolated from a biological sample of a subject. The biological sample can contain any T cells including a sample from the subject. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. Preferably, the subject is human.

[0089] T cells can be obtained from a variety of sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, and tumors. In some embodiments, the T cells can be obtained from a subject having HIV to be treated, i.e., autologous T cells from the subject to be treated. In certain embodiments, the T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art such as Ficoll separation. In some embodiments, cells from the circulating blood of an individual can be obtained by apheresis or leukapheresis. Apheresis products typically contain lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing steps. In one embodiment, the cells can be washed with phosphate buffered saline (PBS). In alternative embodiments, the wash solution can lack calcium, lack magnesium, or lack many or all divalent cations. After washing, the cells can be resuspended in a variety of biocompatible buffers such as, for example, Ca-free, Mg-free PBS. Alternatively, unwanted components of the apheresis sample can be removed and the cells can be resuspended directly in culture medium.

[0090] In another embodiment, T cells can be isolated from peripheral blood by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL gradient. Alternatively, T cells can be isolated from umbilical cord. In any case, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

[0091] In some embodiments, the isolated T cells can include CD4+ T cells and / or CD8+ T cells. CD4 T cells and / or CD8 T cells (CD4 / CD8 T cells) can be isolated from a biological sample by positive or negative selection. Negative selection can be achieved using a combination of antibodies directed against surface markers specific to the negatively selected cells. One method is negative magnetic immunoadhesion or cell sorting and / or selection by flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0092] In the case of isolation of a desired cell population by positive or negative selection, the concentrations of cells and surfaces (e.g., particles such as beads) may vary. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In another embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, more than 100 million cells / ml is used. In yet another embodiment, cell concentrations of 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 50 million cells / ml are used. In yet another embodiment, cell concentrations from 7,500, 8,000, 8,500, 9,000, 95 million, or 100 million cells / ml are used. In yet another embodiment, concentrations of 125 million or 150 million cells / ml can be used. Using high concentrations can lead to increased cell yield, cell activation, and cell proliferation.

[0093] Following the isolation of T cells from a biological sample, in step 20, the isolated CD4 / CD8 T cells can be activated and / or proliferated by any preferred method known in the Art. In embodiments of the present invention, the T cells are activated and the number of T cells is increased in the presence of one or more nonspecific T cell stimuli (e.g., anti-CD3 and anti-CD28) and / or one or more cytokines, cytokines (e.g., IL-1b, IL-2, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12, IL-15, IL-17, IL-21, IL-22, IL-23, IL-35, TGF-β, IFNα, IFNγ, TNFα), recombinant proteins, costimulatory molecules, lectins, ionophores, synthetic molecules, antigen-presenting cells (APCs), artificial APCs, or feeders. In some embodiments, CD4 / CD8 T cells can be activated, and the number of T cells can be increased by physically contacting the T cells with one or more nonspecific T cell stimuli and / or one or more cytokines. Any one or more nonspecific T cell stimuli can be used in the method of the present invention. Examples of nonspecific T cell stimuli include anti-CD3 antibodies and anti-CD28 antibodies. In some embodiments, the nonspecific T cell stimuli may be anti-CD3 antibodies and anti-CD28 antibodies conjugated to beads. Any one or more cytokines can be used in the method of the present invention. Exemplary cytokines include interleukin (IL)-2, IL-7, IL-21, and IL-15.

[0094] Following the activation and / or proliferation of isolated CD4 / CD8 T cells, in step 30, the CD4 / CD8 T cells undergo intermediate co-expression of CD45RA and CD45RO (CD45RA) using, for example, flow cytometry. int CD45RO intThe cells may be isolated or sorted into an enriched population of CD4 / CD8 T cells characterized by ). This method may include sorting the cells by any preferred method. In some embodiments, sorting is performed using flow cytometry. Flow cytometry can be performed using any preferred method known in the art. Flow cytometry can use any preferred antibody and stain. In some embodiments, flow cytometry is multicolor flow cytometry.

[0095] CD45RA produced by the process described herein int CD45RO int The enriched population of CD4 / C8 T cells exhibiting the phenotype is CD45RA. int CD45RO int CD4 / C8 T cells having the above may be included as the majority of cell types. In some embodiments, the process described herein is CD45RA int CD45RO intAt least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, at least about 90%, at least about 89%, at least about 88%, at least about 87%, at least about 86%, at least about 85%, at least about 84%, at least about 83%, at least about 82%, at least about 81%, at least about 80%, at least about 79%, at least about 78%, at least about 77%, at least about 76%, at least about 75%, and less This produces a cell culture and / or cell population containing at least 74%, at least 73%, at least 72%, at least 71%, at least 70%, at least 69%, at least 68%, at least 67%, at least 66%, at least 65%, at least 64%, at least 63%, at least 62%, at least 61%, at least 60%, at least 59%, at least 58%, at least 57%, at least 56%, at least 55%, at least 54%, at least 53%, at least 52%, at least 51%, or at least 50% CD4 / C8 T cells. In a preferred embodiment, the cells in the cell culture or cell population include human cells.

[0096] CD45RA int CD45RO int Long-lived CD4 / CD8 T cells exhibiting this phenotype may also be characterized by the expression of other cell surface markers, such as CD45RA. int CD45RO int Isolated CD4 / CD8 T cells exhibiting the phenotype may express at least one of CD95, CD127, or CD27. In another embodiment, CD45RA int CD45RO int CD4 / CD8 T cells exhibiting the phenotype can further express 4-1BB intermediately and, in some cases, OX40.

[0097] In another embodiment, CD45RA int CD45ROint The isolated CD4 / CD8 T cells having the phenotype can further express at least one, at least two, at least three, at least four, at least five or more of IL17RA, CD5, IL2RG, IGF2R, SLC38A1, IL7R, SLC44A2, SLC2A3, CD96, CD44, CD6, CCR4, IL4R, or SLC12A7.

[0098] In some embodiments, the isolated CD4 / CD8 T cells are CD45RA int CD45RO int and can have the CD95+CD127+CD27+ phenotype. In other embodiments, the isolated T cells are CD45RA int CD45RO int and can have the CD95+CD127+CD27+IL7R+CD44+SCL38A1+IL2RG+CD6+CD5+ phenotype.

[0099] In some embodiments, before and / or after isolating or sorting the CD4 / C8 T cells having the CD45RA int CD45RO int phenotype, the isolated CD4-CD8 T cells having the CD45RA int CD45RO int phenotype can be enriched by culturing the isolated CD4 / CD8 T cells in a cell culture medium containing a small amount of IL-7 and / or IL-15. Activated CD4 / CD8 T cells cultured under low IL-7 / IL-15 conditions (e.g., a concentration of IL7 / IL15 less than 10 ng / ml) can promote or form a concentrated population of CD4 / C8 T cells having the CD45RA int CD45RO int phenotype as compared to activated CD4 / CD8 T cells cultured under high IL-7 / IL-15 conditions (e.g., a concentration of IL-7 / IL-15 greater than 10 ng / ml).

[0100] In some embodiments, the culture medium can contain IL-7 and / or IL-15 at a concentration of, for example, less than about 100 ng / ml, less than about 95 ng / ml, less than about 90 ng / ml, less than about 85 ng / ml, less than about 80 ng / ml, less than about 75 ng / ml, less than about 70 ng / ml, less than about 65 ng / ml, less than about 60 ng / ml, less than about 55 ng / ml, less than about 50 ng / ml, less than about 45 ng / ml, less than about 40 ng / ml, less than about 35 ng / ml, less than about 30 ng / ml, less than about 25 ng / ml, less than about 20 ng / ml, less than about 15 ng / ml, less than about 10 ng / ml, less than about 5 ng / ml, less than about 4 ng / ml, less than about 3 ng / ml, less than about 2 ng / ml, or about 1 ng / ml.

[0101] Using the low IL-7 / IL-15 concentration culture medium described herein, a cell population or cell culture can be enriched at least about 2-fold to about 1000-fold in CD4 / C8 T cells having a CD45RA int CD45RO int phenotype content as compared to an untreated cell population or cell culture. In some embodiments, CD4 / C8 T cells having a CD45RA int CD45RO int phenotype can be enriched at least about 5- to about 500-fold as compared to an untreated cell population or cell culture. In other embodiments, CD4 / C8 T cells having a CD45RA int CD45RO int phenotype can be enriched at least about 10- to about 200-fold as compared to an untreated cell population or cell culture. In still other embodiments, CD4 / C8 T cells having a CD45RA int CD45RO int phenotype can be enriched at least about 20- to about 100-fold as compared to an untreated cell population or cell culture. In still other embodiments, CD4 / C8 T cells having a CD45RA int CD45RO int phenotype can be enriched at least about 40- to about 80-fold as compared to an untreated cell population or cell culture. In certain embodiments, CD45RA int CD45ROint CD4 / C8 T cells exhibiting the phenotype can be enriched at least approximately 2 to 20 times compared to an untreated cell population or cell culture.

[0102] In some embodiments, once isolated or sorted, CD4 / CD8 T cells become CD45RA int CD45RO int To maintain the phenotype, it can be cultured in a culture medium containing TGFβ / IL-1β. CD45RA int CD45RO int Addition of TGFβ and / or IL-1β to phenotypic CD4 / CD8 cells is used before administration to the target CD45RA. int CD45RO int This could lead to the maintenance of the phenotype.

[0103] In some embodiments, the method may further involve genetically modifying CD4 / CD8 T cells before or after activation and / or isolation so that they lack functional CCR5 and / or CXCR4 HIV co-receptors. When HIV infects human T cells, it enters the cell by relying on the association of the T cell receptor CD4 with one of two co-receptors, the chemokine receptor CCR5 or CXCR4. A native CCR5 variant ("CCR5-Δ32") has been identified in human populations that appear to be resistant to HIV infection, particularly in a homozygous state. Therefore, to prevent HIV from infecting T cells and ultimately leading to T cell death and impaired immune function in HIV-infected patients, cells can be made virus-resistant by disrupting one or both of the co-receptors (see U.S. Patent No. 7,951,925). Clinical trials are currently underway in which T cells from HIV patients are edited ex vivo at the CCR5 locus to knock out the CCR5 gene. These cells are then reintroduced into patients to treat HIV.

[0104] In some embodiments, CD4 / CD8 T cells are CD45RA int CD45RO intBefore isolating a population of phenotypic CD4 / CD8 T cells from isolated T cells, the CD4 / CD8 T cells may be genetically modified to lack functional CCR5 and / or CXCR4 HIV co-receptors. In another embodiment, CD45RA int CD45RO int A population of CD4 / CD8 T cells exhibiting the phenotype was isolated from isolated T cells and then subjected to CD45RA int CD45RO int A population of phenotypic CD4 / CD8 T cells may be genetically modified to lack functional CCR5 and / or CXCR4 HIV co-receptors.

[0105] In some embodiments, gene modification or genome editing of CD4 / CD8 T cells may be carried out by transduction, transfection, or electroporation. Transduction may be carried out by electroporation or transfection with lentiviruses, γ-, α-retroviruses or adenoviruses, or nucleic acids (DNA, mRNA, miRNA, antagomyl, ODN), proteins, site-specific nucleases (zinc finger nucleases, TALEN, CRISP / R), self-replicating RNA viruses (e.g., equine encephalopathy virus), or integrated knockout lentiviral vectors.

[0106] In some embodiments, CD45RA int CD45RO intphenotypic CD4 / CD8 T cells and / or CD4 / CD8 T cells can be genetically modified by genome editing using engineered nucleases. Genome editing is the process of inserting, deleting, or modifying genomic sequences using sequence-specific nucleases. Currently, several methods of genome editing exist, including meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), and the CRISPR-Cas system. These nucleases induce double-strand DNA breaks, which can then be repaired by either non-homologous end joining (NHEJ) or homology-dependent repair (HDR), allowing for gene insertion or modification using a template homologous to the DNA surrounding the double-strand break. Traditionally, genome editing is performed by transfecting or transducing cells with RNA or DNA, which then produce proteins, and in the case of the CRISPR-Cas system, the guide RNA required for genome editing.

[0107] For example, double-strand breaks (DSBs) can be created by site-specific nucleases such as zinc finger nucleases (ZFNs) or TAL effector domain nucleases (TALENs). See, for example, Urnov et al. (2010) Nature 435(7042):646-51, U.S. Patents 8,586,526, 6,534,261, 6,599,692, 6,503,717, 6,689,558, 7,067,317, and 7,262,054 (their disclosures are incorporated by reference in their entirety for all purposes).

[0108] Another nuclease system, known as the CRISPR / Cas system, involves the use of a so-called adaptive immune system found in bacteria and archaea. The CRISPR / Cas system is found in 40% of bacteria and 90% of archaea, with varying complexities. See, for example, U.S. Patent No. 8,697,359. CRISPR loci (clustered, regularly spaced short palindromic repeats) are regions within an organism's genome where short segments of foreign DNA are integrated between short, repeating palindromic sequences. These loci are transcribed, and the RNA transcript ("pre-crRNA") is processed into short CRISPR RNA (crRNA). There are three types of CRISPR / Cas systems, all of which incorporate these RNAs and proteins known as "Cas" proteins (CRISPR-associated). Both types I and III possess a Cas endonuclease that processes pre-crRNA, and once fully processed into crRNA, assemble a multi-Cas protein complex capable of cleaving nucleic acids complementary to the crRNA.

[0109] In the type II system, crRNA is produced using a different mechanism in which a transactivating RNA (tracrRNA) complementary to the repeat sequence of pre-crRNA is triggered by double-strand specific RNase III processing in the presence of the Cas9 protein. Cas9 can then cleave target DNA complementary to the mature crRNA, but cleavage by Cas9 depends on both base pairing between the crRNA and the target DNA, and the presence of a short motif within the crRNA called a PAM sequence (protospacer adjacent motif). Furthermore, the tracrRNA must be present to base pair with the crRNA at its 3' end, and this association triggers Cas9 activity.

[0110] The Cas9 protein has at least two nuclease domains; one is similar to an HNH endonuclease domain, and the other is similar to a Ruv endonuclease domain. The HNH-type domain appears to be responsible for cleaving DNA strands complementary to the crRNA, while the Ruv domain cleaves non-complementary strands.

[0111] The requirement for a crRNA-tracrRNA complex can be avoided by using an engineered “single guide RNA” (sgRNA) containing a hairpin typically formed by crRNA and tracrRNA annealing (see Jinek et al (2012) Science 337:816 and Cong et al (2013) Sciencexpress / 10.1126 / science.1231143). In S. pyrogene, once a double-stranded RNA:DNA heterodimer is formed between Cas-associated RNA and target DNA, an engineered tracrRNA:crRNA fusion or sgRNA guides Cas9 to cleave the target DNA. This system, containing the Cas9 protein and an engineered sgRNA with a PAM sequence, has been used for RNA-guided genome editing (see Ramalingam ibid) and is useful for in vivo zebrafish embryo genome editing with editing efficiencies similar to ZFNs and TALENs (see Hwang et al (2013) Nature Biotechnology 31(3):227).

[0112] Certain nucleases can also be engineered to insert peptide fusion inhibitors into the HIV receptor to prevent HIV infection in T cells (see co-owned U.S. Patent Publication No. 2012 / 0093787), examples of such peptide fusion inhibitors are C34 or Fuzeon. Similarly, HIV can be treated by using engineered nucleases to insert anti-HIV transgenes into safe harbor loci within the cell to fight the virus. Examples of such HIV genes can be selected from the group consisting of sequences encoding zinc finger transcription factors that suppress HIV polyproteins, sequences encoding zinc finger transcription factors that suppress the expression of HIV receptors, CCR5 ribozymes, siRNA sequences targeted to HIV polyproteins, sequences encoding the Trim5α restriction factor, sequences encoding the APOBEC3G restriction factor, sequences encoding the RevM10 protein, sequences encoding C46, other anti-HIV genes, suicide cassettes, and combinations thereof. Thus, the methods and compositions of the invention can be used to treat or prevent HIV with a CRISPR / Cas system in which a single guide RNA contains sequences targeting the CCR5 or CXCR4 gene for the integration of a suitable anti-HIV transgene.

[0113] In some embodiments, genome editing may involve cleavage by a site-specific nuclease for targeted insertion into a selected genomic locus (see, for example, U.S. Patent No. 7,888,121, co-owned). Nucleases specific to the target gene can be used to ensure that the transgene construct is inserted either by homology-directed repair (HDR) or by end capture during a non-homologous end junction (NHEJ) driven process. Target loci include the AAVS1, HPRT, and CCR5 genes in human cells, as well as “safe harbor” loci such as Rosa26 in mouse cells (see, for example, U.S. Patents No. 7,888,121, 7,972,854, 7,914,796, 7,951,925, 8,110,379, 8,409,861, 8,586,526, and U.S. Patent Publication No. 2003 / 02324). See issues 10, 2005 / 0208489, 2005 / 0026157, 2006 / 0063231, 2008 / 0159996, 2010 / 00218264, 2012 / 0017290, 2011 / 0265198, 2013 / 0137104, 2013 / 0122591, 2013 / 0177983, and 2013 / 0177960). Nuclease-mediated integration allows for precise transgene positioning while minimizing the risk of gene silencing or activation of nearby oncogenes, thus offering prospects for improved transgene expression, safety, and expression durability compared to conventional integration approaches that rely on random transgene integration.

[0114] In addition to the insertion methods described above, genome editing can also include gene knockout. When a donor nucleic acid is unavailable, cells with a broken genome repair the break using the error-prone NHEJ pathway. This process often involves the addition or deletion of nucleotides ("indels") during the repair process, which can lead to the introduction of missense or nonsense mutations at the target site.

[0115] For example, CCR5-specific zinc finger nucleases have been used in Phase I / II trials to create non-functional CCR5 receptors in T cells and thus prevent HIV infection (see U.S. Patent No. 7,951,925). These cells are then reintroduced into patients to treat HIV. Thus, the methods and compositions of the present invention can be used to disrupt the CCR5 allele in a CRISPR / Cas system, and the single guide RNA contains a sequence that targets the human CCR5 gene (chr3:46411633-46417697), particularly in or near the exon region (chr3:46414394-46415452).

[0116] One particularly preferred region for targeting the CCR5 gene for knockout is the region near the δ-32 mutation region (chr3:46414923–46415020 or nearby). Another particularly preferred region is around chr3:46414522–46414643, which encodes part of the second extracellular loop of the CCR5 protein. The ATG protein translation start site or a region near it (chr3:46414347–46414466 or nearby) is also particularly preferred for genomic modifications such as the fusion of the C34 peptide to the N-terminus of CCR5 by targeted integration of anti-HIV therapy.

[0117] Similar research is underway in animal models of CXCR4-dependent HIV, where CXCR4 is selectively disrupted or disrupted in parallel with CCR5 to prevent HIV infection of T cells (see U.S. Patent Publication 2010 / 0291048). Thus, the CXCR4 allele can be disrupted in a CRISPR / Cas system using the methods and compositions described herein, and the single guide RNA contains a sequence that targets the human CXCR4 gene (chr2:136871919-136875725), particularly in or near the region surrounding the exon 2 region (chr2:136872439-136873482) and the small exon 1 region (chr2:136875616-136875630). One preferred region for targeting the CXCR4 gene for knockout is chr2:136872863-136872982 or a nearby region, which is an analogue to the δ-32 mutant region of the CCR5 gene. Particularly preferred are chr2:136875540-136875687 or a nearby region near the ATG protein translation start site in exon 1, and chr2:136873389-136873558 or a nearby region near the splicing site in exon 2, which are particularly preferred for gene modifications such as the fusion of the C34 peptide to the N-terminus of CXCR4 by targeted integration for anti-HIV therapy. Thus, sgRNAs can be designed to bind to sequences at any location on the CCR5 or CXCR4 locus, including but not limited to sequences of one or more of these preferred target regions.

[0118] Compositions comprising nucleases, polynucleotides encoding these nucleases, donor polynucleotides, and proteins and / or polynucleotides described herein are ex vivo and transmitted by any preferred means to CD4 / CD8 T cells and / or CD45RA int CD45RO int It can be delivered to phenotypic CD4 / CD8 T cells.

[0119] Methods for delivering the nucleases described herein are described, for example, in U.S. Patents 6,453,242, 6,503,717, 6,534,261, 6,599,692, 6,607,882, 6,689,558, 6,824,978, 6,933,113, 6,979,539, 7,013,219, and 7,163,824, all of which are incorporated herein by reference in their entirety.

[0120] The nucleases and / or donor constructs described herein may also be delivered using vectors containing sequences encoding one or more of the CRISPR / Cas systems. Any vector system may be used, including but not limited to plasmid vectors, DNA minicircles, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, and combinations thereof. See also U.S. Patents 6,534,261, 6,607,882, 6,824,978, 6,933,113, 6,979,539, 7,013,219, and 7,163,824, as well as U.S. Patent Publication 2014 / 0335063 (which are incorporated herein in their entirety by reference). Furthermore, it will be apparent that any of these vectors may contain one or more of the sequences required for therapeutic purposes. Therefore, when one or more nucleases and donor constructs are introduced into cells, the nucleases and / or donor polynucleotides may be carried on the same vector or on different vectors. If multiple vectors are used, each vector may contain sequences encoding one or more nucleases and / or donor constructs.

[0121] Conventional viral and nonviral-based gene transfer methods can be used to introduce nuclease-encoding nucleic acids and donor constructs into cells (e.g., mammalian cells) and target tissues. Nonviral vector delivery systems include DNA plasmids, DNA minicircles, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes or poloxamers. Viral vector delivery systems include DNA and RNA viruses that have either episomes or integrated genomes after delivery to cells. For reviews of gene therapy procedures, see Anderson, Science 256:808-813 (1992), Nabel & Feigner, TIBTECH 11:211-217 (1993), Mitani & Caskey, TIBTECH 11:162-166 (1993), Dillon, TIBTECH 11:167-175 (1993), Miller, Nature 357:455-460 (1992), Van Brunt, Biotechnology 6(10):1149-1154 (1988), Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995), Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995), and Haddada et al., in Current Topics. See in Microbiology and Immunology, Doerfler and Bohm (eds.) (1995), and Yu et al., Gene Therapy 1:13-26 (1994).

[0122] Nonviral methods of nucleic acid delivery include electroporation, lipofection, microinjection, particulate guns, virosomes, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, naked RNA, capped RNA, artificial virions, and drug-enhanced uptake of DNA. For example, sonoporation using the Sonitron 2000 system (Rich-Mar) can also be used for nucleic acid delivery.

[0123] Additional exemplary nucleic acid delivery systems include those offered by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.), and Copernicus Therapeutics Inc. (see, for example, U.S. Patent No. 6,008,336). Lipofection is described, for example, in U.S. Patents No. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include those described in Feigner, WO91 / 17424 and WO91 / 16024.

[0124] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those skilled in the art (e.g., Crystal, Science 270:404-410 (1995), Blaese et al., Cancer Gene Ther. 2:291-297 (1995), Behr et al., Bioconjugate Chem. 5:382-389 (1994), Remy et al., Bioconjugate Chem. 5:647-654 (1994), Gao et al., Gene Therapy 2:710-722 (1995), Ahmad et al., Cancer See Res.52:4817-4820 (1992), U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0125] Additional delivery methods include packaging nucleic acids to be delivered to EnGeneIC delivery vehicles (EDVs). These EDVs are specifically delivered to target tissues using bispecific antibodies, where one arm of the antibody is specific to the target tissue and the other arm is specific to the EDV. The antibody carries the EDV to the target cell surface, and then the EDV is delivered into the cell by endocytosis. Once inside the cell, the contents are released (see MacDiarmid et al. (2009) Nature Biotechnology 27(7):643).

[0126] The use of RNA or DNA virus-based systems for the delivery of nucleic acids encoding modified CRISPR / Cas systems leverages highly evolved processes to target viruses to specific cells in the body and transport the viral payload to the nucleus. Viral vectors can be administered directly to the target (in vivo) or used to process cells in vitro, and modified cells can be administered to the target (ex vivo). Conventional virus-based systems for the delivery of CRISPR / Cas systems include, but are not limited to, retroviral, lentiviral, adenovirus, adeno-associated, vaccinia, and herpes simplex virus vectors for gene transfer. Integration into the host genome is possible with retroviral, lentiviral, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgene. Furthermore, high transduction efficiencies have been observed in many different cell types and target tissues.

[0127] The tropism of retroviruses can be modified by incorporating foreign envelope proteins to expand the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeat sequences with the ability to package foreign sequences up to 6–10 kb. The smallest cis-acting LTRs are sufficient for vector replication and packaging, and are then used to integrate therapeutic genes into target cells to provide permanent transgene expression. Widely used retroviral vectors include those based on mouse leukemia virus (MuLV), gibbon leukemia virus (GaLV), Simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, for example, Buchscher et al., J.Virol.66:2731-2739 (1992), Johann et al., J.Virol.66:1635-1640 (1992), Sommerfelt et al., Virol.176:58-59 (1990), Wilson et al., J.Virol.63:2374-2378 (1989), Miller et al., J.Virol.65:2220-2224 (1991), PCT / US94 / 05700).

[0128] For applications where transient expression is preferred, adenovirus-based systems can be used. Adenovirus-based vectors enable very high transduction efficiency in many cell types and do not require cell division. High titers and high levels of expression have been obtained using such vectors. These vectors can be produced in large quantities using relatively simple systems. Adeno-associated virus ("AAV") vectors are also used to transduce cells with target nucleic acids, for example, in the in vitro production of nucleic acids and peptides, as well as for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987), U.S. Patent No. 4,797,368, WO93 / 24641, Kotin, Human Gene Therapy 5:793-801 (1994), Muzyczka, J. Clin. Invest. 94:1351 (1994)). Constructions of recombinant AAV vectors have been described in numerous publications, including U.S. Patent No. 5,173,414, Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985), Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984), Hermonat & Muzyczka, PNAS 81:6466-6470 (1984), and Samulski et al., J. Virol. 63:03822-3828 (1989).

[0129] At least six viral vector approaches are currently available for gene transfer in clinical trials, utilizing approaches that involve supplementing defective vectors with genes inserted into helper cell lines to generate transduction agents.

[0130] pLASN and MFG-S are examples of retroviral vectors used in clinical trials (Dunbar et al., Blood 85:3048-305 (1995), Kohn et al., Nat. Med. 1:1017-102 (1995), Malech et al., PNAS 94:22 12133-12138 (1997)). PA317 / pLASN was the first therapeutic vector used in gene therapy trials (Blaese et al., Science 270:475-480 (1995)). Transduction efficiencies of over 50% have been observed with the MFG-S packaged vector (Ellem et al., Immunol Immunother. 44(1):10-20 (1997), Dranoff et al., Hum. Gene Ther. 1:111-2 (1997).

[0131] Recombinant adeno-associated virus vectors (rAAV) are a promising alternative gene delivery system based on defective, non-pathogenic parvovirus adeno-associated virus type 2. All vectors are derived from plasmids containing only the inverted terminal repeat of 145 base pairs (bp) of AAV adjacent to the transgene expression cassette. Efficient gene transfer and stable transgene delivery, resulting from integration into the genome of transduced cells, are key features of this vector system. (Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther. 9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, and AAVrh10, as well as all their variants, can also be used according to the present invention.

[0132] Replication-deficient recombinant adenovirus vectors (Ad) are produced at high titers and readily infect many different cell types. Most adenovirus vectors are engineered so that the transgene replaces the Ad E1a, E1b, and / or E3 genes, and the replication-deficient vector then proliferates in human cells that supply the function of the gene deleted in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing differentiated cells such as those found in the liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity. Examples of Ad vector use in clinical trials include polynucleotide therapy for antitumor immunity via intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al., Infection 24:1 5-10 (1996), Sterman et al., Hum.Gene Ther. 9:7 1083-1089 (1998), Welsh et al., Hum.Gene Ther. 2:205-18 (1995), Alvarez et al., Hum.Gene Ther. 5:597-613 (1997), Topf et al., Gene Ther. 5:507-513 (1998), and Sterman et al., Hum.Gene Ther. 7:1083-1089 (1998).

[0133] Packaging cells are used to form viral particles that can infect host cells. Such cells include 293 cells for packaging adenoviruses and psi2 or PA317 cells for packaging retroviruses. Viral vectors used in gene therapy are typically produced by producer cell lines that package nucleic acid vectors into viral particles. The vectors usually contain the minimum viral sequences necessary for packaging and subsequent integration into the host (if applicable), with other viral sequences replaced by expression cassettes encoding the proteins to be expressed. Missing viral functions are supplied trans by the packaging cell line. For example, AAV vectors used in gene therapy typically contain only inverted end repeat (ITR) sequences from the AAV genome, which are necessary for packaging and integration into the host genome. The viral DNA is packaged into a cell line that contains helper plasmids encoding other AAV genes, i.e., rep and cap, but lacking the ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus facilitates the replication of the AAV vector and the expression of AAV genes from the helper plasmids. Because there is no ITR sequence, the helper plasmid is not packaged in large quantities. Adenovirus contamination can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV.

[0134] The gene editing vector is used in CD4 / CD8 T cells and / or CD45RA int CD45RO intThe gene-editing vector can be delivered ex vivo to phenotypic CD4 / CD8 T cells, followed by re-implantation of the cells into the patient, usually after selection of the vector-incorporated cells. Formulations containing gene-editing vectors for ex vivo administration may include suspensions in liquids or emulsified liquids. The active ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and combinations thereof. Furthermore, the formulation may contain small amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, or other reagents that enhance the efficacy of the pharmaceutical composition.

[0135] CD45RA produced by the method described herein int CD45RO int Selected, enriched populations of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells possessing a specific phenotype may be incorporated into compositions, such as pharmaceutical compositions, for treating HIV infection in a subject. The composition may also include a pharmaceutically acceptable carrier. With respect to the pharmaceutical composition, the carrier may be one of those conventionally used for cell administration. Such pharmaceutically acceptable carriers are well known to those skilled in the art and are generally readily available. It is preferable that the pharmaceutically acceptable carrier does not have adverse side effects or toxicity under the conditions of use.

[0136] The composition can be prepared in unit dosage forms for administration to the target. The amount and timing of administration are left to the discretion of the clinician treating the patient to achieve the desired outcome. The composition can be formulated for systemic administration (e.g., intravenous administration) or local administration (e.g., intratumoral administration). In one example, CD45RA int CD45RO int Enriched populations of phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells are formulated for parenteral administration, such as intravenous administration. CD45RA int CD45RO intA composition containing an enriched population of phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells can be used, for example, for the treatment of HIV in a subject.

[0137] CD45RA is provided as a composition for administration, on a pharmaceutically acceptable carrier such as an aqueous carrier. int CD45RO int The formulations may include a solution of enriched populations of phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells. Various aqueous carriers, such as buffered saline, can be used. These solutions are sterile and generally free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable adjuvants necessary to approximate physiological conditions, such as pH adjusters and buffers, toxicity modifiers, and adjuvants, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The number of cells or CD45RA in these formulations may be considered. int CD45RO int The concentrations of phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, etc., according to the specific dosage form chosen and the needs of the target. Practical methods for preparing such dosage forms for use in gene therapy, immunotherapy, and / or cell therapy will be known or evident to those skilled in the art.

[0138] In one embodiment, CD45RA int CD45RO int A concentrated population of phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells can be added to an infusion bag containing 0.9% sodium chloride and USP, and administered at a dose of 0.5-15 mg / kg of body weight, if necessary. CD45RA int CD45RO intA population of enriched CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells exhibiting the phenotype can be administered by slow infusion rather than intravenous push or bolus. In one example, a higher loading dose was administered, followed by a lower maintenance dose.

[0139] The dose administered, for example, CD45RA int CD45RO int The number of phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells should be sufficient, for example, to produce a therapeutic or prophylactic response in a subject or animal over a reasonable timeframe. For example, CD45RA int CD45RO int The number of phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells should be sufficient to treat HIV for a period of approximately 6 months, 1 year, 2 years, 3 years, and 4 years or more from the time of administration. In certain embodiments, the period may be even longer. CD45RA int CD45RO int The number of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells exhibiting the phenotype is, for example, CD45RA. int CD45RO int The efficacy of the phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells and the condition of the animal (e.g., human), as well as the body weight of the animal being treated (e.g., human), will be determined.

[0140] CD45RA int CD45RO int The number of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells with the phenotype is also CD45RA int CD45RO intThe choice of CD45RA will also depend on the presence, nature, and severity of any adverse side effects that may occur with the administration of an enriched population of phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells. Typically, the attending physician will consider various factors such as age, weight, general health, diet, sex, route of administration, and severity of the condition being treated when deciding which CD45RA to use to treat each individual patient. int CD45RO int The number of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells possessing the phenotype of the present invention will be determined. For example, without intending to limit the present invention, CD45RA int CD45RO int The number of phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells is approximately 10 × 10 per injection. 4 ~About 10×10 11 Cells, approximately 10 x 10 per injection 5 Cells ~ approx. 10×10 9 Cells, or 10 x 10 per injection 7 ~About 10×10 9 It could be a cell.

[0141] CD45RA int CD45RO int Phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells are intended to be used in methods for treating or preventing HIV infection in subjects requiring such treatment. In this regard, methods for treating or preventing HIV infection in subjects are described herein as CD45RA int CD45RO int This may include administering to a subject an enriched population of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells that may possess a phenotypic trait, in a dose effective in treating or preventing HIV in the subject.

[0142] In some embodiments, administration of a composition or enriched T cell population to a subject having HIV can promote at least one of the following: a sustained increase in the absolute CD4 cell count in the subject, restoration of HIV-specific T cell immunity, and substantial decay of the HIV reservoir. In some embodiments, the subject is receiving and / or continuing to receive antiretroviral therapy.

[0143] CD45RA int CD45RO int The administered CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells, which possess the phenotype, may be allogeneic or autologous to the host or target. Preferably, the cells are autologous to the target.

[0144] In some embodiments, CD45RA int CD45RO int Phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells can be administered in combination with activators of latent HIV expression. Several activators of latent HIV expression can be used in the compositions and methods described herein. For example, activators of latent HIV expression may include, but are not limited to, histone deacetylase (HDAC) inhibitors and protein kinase C agonists.

[0145] HDAC inhibitors have been demonstrated to induce transcriptional activation of the HIV-1 promoter. HDAC inhibitors can be any molecule that reduces the activity of histone deacetylase. These include proteins, peptides, DNA molecules (including antisense), RNA molecules (including iRNA agents and antisense), and small molecules. In some embodiments, the HDAC inhibitor is a small interfering RNA (siRNA), e.g., si / shRNA directed to HDAC1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. Non-limiting examples of such HDAC inhibitors are listed below. It is understood that HDAC inhibitors include any salts, crystalline structures, amorphous structures, hydrates, derivatives, metabolites, stereoisomers, structural isomers, and prodrugs of the HDAC inhibitors described herein.

[0146] In some embodiments, the HDAC inhibitor may contain short-chain fatty acids (e.g., sodium butyrate, isovaleric acid, valeric acid, 4-phenylbutyric acid (4-PBA), phenylbutyric acid (PB), propionic acid, butylamide, isobutylamide, phenylacetic acid, 3-bromopropionate, triptyline, valproic acid (Vpa), valproate, semisodium valproate, and pivaloyloxymethyl butyrate (PIVANEX)).

[0147] In other embodiments, the HDAC inhibitor is a hydroxamic acid derivative (e.g., suberoylanilide hydroxamic acid (SAHA, vorinostat), trichostatin analogs such as trichostatin A (TSA) and trichostatin C, m-carboxycinnamate bishydroxamic acid (CBHA), pyroxamide, salicylic acid bishydroxamic acid, suberoylbishydroxamic acid (SBHA), azeraate bishydroxamic acid (ABHA), azeraate-1-hydroxamic acid-9-anilide (AAHA), 6-(3-chlorophenylureido)caproate hydroxamic acid (3Cl-UCHA), oxamfratin [(2E)-5-[3-[(phenylsulfonyl)amino]phenyl]-pento-2-en-4-inohydroxamic acid], A-161906 It may include Scriptaid, PXD-101 (Prolifix), LAQ-824, CHAP, MW2796, MW2996; or any of the hydroxamic acids disclosed in U.S. Patents 5,369,108, 5,932,616, 5,700,811, 6,087,367, and 6,511,990. In certain embodiments, the HDAC inhibitor is a SAHA.

[0148] In yet another embodiment, the HDAC inhibitor may include benzamide derivatives (e.g., CI-994;MS-275[N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxycarbonyl)aminomethyl]benzamide] and 3'-amino derivatives of MS-275).

[0149] In further embodiments, HDAC inhibitors may include cyclic peptides (e.g., trapoxin A (TPX)-cyclic tetrapeptide (cyclo-(L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8-oxo-9,10-epoxydecanoyl)), FR901228 (FK228, depsipeptide), FR225497 cyclic tetrapeptide, apicidine cyclic tetrapeptide [cyclo(NO-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)]), apicidine Ia, apicidine Ib, apicidine Ic, apicidine IIa, and apicidine IIb, CHAP, HC toxin cyclic tetrapeptide, WF27082 cyclic tetrapeptide, and chlamydosin.

[0150] Additional HDAC inhibitors may include natural products such as samaprines and depdesine, electrophilic ketone derivatives such as trifluoromethyl ketones, α-ketoamides such as N-methyl-α-ketoamide, LSD1 polypeptides, TNF-alpha (TNFα), inducible transcription factor NF-AT (nuclear factor of activated T cells), and anti-IκBα or IκBε agents.

[0151] CD45RA int CD45RO int Phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells can be administered alone or in combination with activators of latent HIV expression described herein to subjects with latent HIV infection, e.g., humans with latent HIV infection. Subjects may include those who have a persistent HIV reservoir despite treatment with antiretroviral therapy (e.g., HAART). Thus, in some embodiments, a therapeutically effective dose is used to significantly reduce the latent HIV reservoir in subjects with latent HIV infection using CD45RA. int CD45RO int This is the quantity of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells exhibiting the phenotype.

[0152] In another embodiment, a therapeutically effective dose of CD45RA int CD45RO int Phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells, along with optionally activators of latent HIV expression, can be administered to subjects in combination with other therapeutic agents, such as components used in HAART or immunotoxins, which are useful in treating HIV infection.

[0153] As described above, CD45RA as described in this specification int CD45RO int A phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T may be combined with one or more additional therapeutic agents useful for treating HIV infection. CD45RA int CD45RO int The range of combinations of phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells with HIV / AIDS antiviral agents, immunomodulators, anti-infective agents, or vaccines is not limited to the list below and will be understood to include, in principle, any combination with any pharmaceutical composition useful for the treatment of AIDS. HIV / AIDS antiviral agents and other drugs will typically be used in these combinations within their conventional dosage ranges and regimens, as reported in the Art.

[0154] Examples of antiviral drugs (but not limited to) include antiviral drug manufacturers (trade name and / or drug name, location), indications (activity): Abacavir GlaxoSmithKline HIV infection, AIDS, ARC, GW 1592(ZIAGEN)(nRTI); 1592U89 Abacavir + GlaxoSmithKline HIV infection, AIDS, ARC (nnRTI); Lamivudine + (TRIZIVIR) Zidovudine Acemanna Carrington Labs ARC (Irving,Tex.) ACH 126443 Achillion Pharm. HIV infection, AIDS, ARC (nucleoside reverse transcriptase inhibitor); Acyclovir Burroughs Wellcome HIV infection, AIDS, ARC, AZT (in combination with AZT) AD-439 Tanox Biosystems HIV infection, AIDS, ARC AD-519 Tanox Biosystems HIV infection, AIDS, ARC Adefovir Dipivoxil Gilead HIV infection, AIDS, ARC GS840 (RTI); AL-721 Ethigen ARC, PGL, HIV positive (Los Angeles, Calif.), AIDS α interferon GlaxoSmithKline Kaposi's sarcoma, HIV (in combination with Retrovir) AMD3100 AnorMed HIV infection, AIDS, ARC (CXCR4 antagonist); Amprenavir GlaxoSmithKline HIV infection, AIDS, 141 W94 (AGENERASE) ARC (PI); GW 141 VX478 (Vertex) Ansamycin Adria Laboratories ARC LM427 (Dublin, Ohio) Erbamont (Stamford, Conn.) antibody (neutralizing); Advanced Biotherapy AIDS, ARC pH unstable α abnormality Concepts (Rockville, Interferon Md.AR177 Aronex Pharm HIV infection, AIDS, ARC Atazanavir (BMS 232632) Bristol-Myers-Squibb HIV infection, AIDS, ARC (ZRIVADA) (PI); β-fluoro-ddA Nat'l Cancer Institute AIDS-related diseases BMS-232623 Bristol-Myers Squibb / HIV infection, AIDS, (CGP-73547) Novartis ARC (PI); BMS-234475 Bristol-Myers Squibb / HIV infection, AIDS, (CGP-61755) Novartis ARC (PI); Caplavillin Pfizer HIV infection, AIDS, (AG-1549, S-1153) ARC (nnRTI); CI-1012 Warner-Lambert HIV-1 infection Zidofovir Gilead Science CMV retinitis, herpes, papillomavirus Curdran sulfate AJI Pharma USA HIV infection, cytomegalovirus immunity, MedImmune CMV retinitis, globin cytovene Syntex, vision-threatening CMV ganciclovir, peripheral CMV retinitis delaviridine, Pharmacia-Upjohn HIV infection, AIDS, (RESCRIPTOR)ARC (nnRTI); dextran sulfate, Ueno Fine Chem.Ind. AIDS, ARC,HIV Ltd. (Osaka, Japan) positive asymptomatic ddC, Hoffman-La Roche HIV infection, AIDS, ARC (zalcitabine, (HIVID) (nRTI); dideoxycytidine ddl, Bristol-Myers Squibb HIV infection, AIDS, ARC; dideoxyinosine (VIDEX) (combination with AZT / d4T) (nRTI) DPC 681 & DPC 684, DuPont HIV infection, AIDS, ARC (PI) DPC 961 & DPC 083, DuPont HIV infection AIDS, ARC (nnRTRI); Embilin Triangle Pharmaceuticals HIV infection, AIDS, ARC (COACTINON) (non-nucleoside reverse transcriptase inhibitor); EL10 Elan Corp, PLC HIV infection (Gainesville, Ga.Efavirenz DuPont HIV infection, AIDS, (DMP 266) (SUSTIVA) ARC (nnRTI); Merck (STOCRIN) Famciclovir Smith Kline Herpes zoster, herpes simplex emtricitabine Triangle Pharmaceuticals HIV infection, AIDS, ARC FTC (COVIRACIL) (nRTI); Emvirin Triangle Pharmaceuticals HIV infection, AIDS, ARC (COACTINON) (non-nucleoside reverse transcriptase inhibitor); HBY097 Hoechst Marion Roussel HIV infection, AIDS, ARC (nnRTI); Hypericin VIMRx Pharm. HIV infection, AIDS, ARC recombinant human; Triton Biosciences AIDS, Kaposi's sarcoma, interferon β (Almeda, Calif.); ARC interferon α-n3 Interferon Sciences ARC, AIDS indinavir; Merck (CRIXIVAN) HIV infection, AIDS, ARC, asymptomatic HIV positive (or in combination with AZT / ddI / ddC) (PI); ISIS 2922 ISIS Pharmaceuticals CMV retinitis JE2147 / AG1776; Agouron HIV infection, AIDS, ARC (PI); KNI-272 Nat'l Cancer Institute HIV-related disease lamivudine; 3TC Glaxo Wellcome HIV infection, AIDS, (EPIVIR)ARC; or with AZT (nRTI); Lobukavir Bristol-Myers Squibb CMV infection; Lopinavir (ABT-378) Abbott HIV infection, AIDS, ARC (PI); Lopinavir + Ritonavir Abbott (KALETRA) HIV infection, AIDS, ARC (ABT-378 / r) (PI); Mozenavir AVID (Camden, NJ))HIV infection, AIDS, ARC (DMP-450) (PI); Nelfinavir Agouron HIV infection, AIDS, (VIRACEPT)ARC (PI); Nevirapine Boeheringer HIV infection, AIDS, Ingleheim ARC (nnRTI); (VIRAMUNE) Novaprene Novaferon Labs, Inc. HIV inhibitor (Akron, Ohio); Pentaphside Trimeris HIV infection, AIDS, ARC T-20 (fusion inhibitor); Peptide T Peninsula Labs AIDS octapeptide (Belmont, Calif.) sequence PRO 542 Progenics HIV infection, AIDS, ARC (adhesion inhibitor); PRO 140 Progenics HIV infection, AIDS, ARC (CCR5 co-receptor inhibitor); Trisodium Astra Pharm. Products, CMV retinitis, HIV infection, Phosphonoformate Inc. Other CMV infections; PNU-140690 Pharmacia Upjohn HIV infection, AIDS, ARC (PI); Probucol Vyrex HIV, AIDS; RBC-CD4 Sheffield Med.Tech HIV, AIDS, (Houston Tex.)ARC; Ritonavir Abbott HIV, AIDS, (ABT-538)(RITONAVIR)ARC(PI); Saquinavir Hoffmann-LaRoche HIV, AIDS, (FORTOVASE)ARC(PI); Stavudine d4T Bristol-Myers Squibb HIV infection, AIDS, ARC didehydrodeoxy-(ZERIT.) (nRTI); Thymidine T-1249 Trimeris HIV infection, AIDS, ARC (fusion inhibitor); TAK-779 Takeda HIV infection, AIDS, ARC (injectable CCR5 receptor antagonist); Tenofovir Gilead (VIREAD) HIV infection, AIDS, ARC (nRTI); Tipranavir (PNU-140690) Boehringer Ingelheim HIV infection, AIDS, ARC(PI);TMC-120&TMC-125 Tibotec HIV infection, AIDS, ARC(nnRTI);TMC-126 Tibotec HIV infection, AIDS, ARC(PI);Valaciclovir GlaxoSmithKline Genital HSV&CMV infection Virazole Viratek / ICN(Costa asymptomatic HIV positive, ribavirin Mesa,Calif.) LAS, ARC; zidovudine; AZT GlaxoSmithKline HIV infection, AIDS, ARC, (RETROVIR) Kaposi's sarcoma, and in combination with other therapies (nRTI); [PI = protease inhibitor, nnRTI = non-nucleoside reverse transcriptase inhibitor, NRTI = nucleoside reverse transcriptase inhibitor].

[0155] Additional medications may be administered individually, sequentially, or as CD45RA int CD45RO int It can be used in combination with phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells. Administration to the target population can be done via the same or different routes of administration, or together with the same pharmaceutical formulation.

[0156] According to this embodiment, CD45RA int CD45RO int CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells possessing phenotypic and latent HIV expression activators can be administered concurrently with any HAART regimen or its components. Current standard treatment with HAART typically involves a combination of at least three nucleoside reverse transcriptase inhibitors, often including protease inhibitors or non-nucleoside reverse transcriptase inhibitors. CD4+ Subjects with low cell counts or high plasma RNA levels may require more aggressive HAART. CD4 + In subjects with relatively normal cell counts and low or unmeasurable plasma HIV RNA levels over a long period (i.e., slow or non-progressive), less aggressive HAART may be required. For antiretrovirally naive subjects treated with an initial antiretroviral regimen, different combinations (or cocktails) of antiretroviral drugs may be used.

[0157] Therefore, in some embodiments, CD45RA int CD45RO int Phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells, and optionally activators of latent HIV expression, can be co-administered to the target group along with a "cocktail" of nucleoside reverse transcriptase inhibitors, non-nucleoside HIV reverse transcriptase inhibitors, and protease inhibitors. For example, CD45RA int CD45RO int Phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells and HDAC inhibitors can be administered concurrently with a cocktail of two nucleoside reverse transcriptase inhibitors (e.g., zidovudine (AZT) and lamivudine (3TC)) and one protease inhibitor (e.g., indinavir (MK-639)). CD45RA int CD45RO int CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells exhibiting the phenotype, as well as optionally activators of latent HIV expression such as HDAC inhibitors, can be simultaneously administered to a cocktail of one nucleoside reverse transcriptase inhibitor (e.g., stabudine (d4T)), one non-nucleoside reverse transcriptase inhibitor (e.g., nevirapine (BI-RG-587)), and one protease inhibitor (e.g., nelfinavir (AG-1343)). Alternatively, CD45RA int CD45RO intPhenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells, along with optionally HDAC inhibitors, can be co-administered with a cocktail of one nucleoside reverse transcriptase inhibitor (e.g., zidovudine (AZT)) and two protease inhibitors (e.g., nelfinavir (AG-1343) and saquinavir (Ro-31-8959)).

[0158] In the context of this invention, concurrent administration is defined as the administration of multiple therapeutic agents in the course of coordinated treatment to achieve improved clinical outcomes. Such concurrent administration may also have the same extent, i.e., may occur during overlapping periods.

[0159] In an additional embodiment, the immunotoxin is CD45RA int CD45RO int It can be co-administered to phenotypic CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells. Examples of immunotoxins are those that target HIV proteins expressed outside the cell, such as viral envelope glycoproteins or parts thereof. The term “immunotoxin” refers to the covalent or non-covalent binding of a toxin to an antibody, such as an anti-HIV envelope glycoprotein antibody. The toxin may be directly linked to the antibody or indirectly linked, for example, via a linker molecule. Toxins can be selected from the group consisting of lysine-A and abrin-A.

[0160] Activation of latent HIV expression (also referred to as reactivation of latent HIV expression) results in the conversion of latent infected cells into productively infected cells. This transition can be measured by any characteristic of active viral infection, such as the production of infectious particles, reverse transcriptase activity, secreted antigens, cell surface antigens, soluble antigens, HIV RNA, and HIV DNA. The methods described herein may optionally include a step of determining or detecting the activation of latent HIV expression. In one embodiment, such a method includes determining or detecting mRNA, for example, HIV mRNA. Other mRNAs, such as Tat mRNA, NF-κB mRNA, NF-AT mRNA, and other mRNAs encoding polypeptides, can also be determined using well-known methods, including but not limited to hybridization and amplification-based assays.

[0161] In another embodiment, an amplification-based assay is used to measure the expression level of HIV genes. In one embodiment, activation of latent HIV expression can be detected by determining the expression level of HIV polypeptides. The expression level of HIV polypeptides can be determined by several methods, including but not limited to affinity capture, mass spectrometry, conventional immunoassays targeting HIV proteins (such as gp120 and reverse transcriptase), PAGE, Western blotting, or HPLC, as further described herein or known to those skilled in the art.

[0162] Detection paradigms that can be used for this purpose include optical methods, electrochemical methods (voltage measurement and amperometry techniques), atomic force microscopy, and radio frequency methods, such as multipolar resonance spectroscopy. In addition to microscopy, examples of both confocal and non-confocal optical methods include the detection of fluorescence, emission, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, resonant microscopy, grating coupler waveguide, or interferometry).

[0163] In some embodiments, global sequencing and 454 pyrosequensing of HIV-based vector constructs and PCR products described herein can be performed to confirm the production and purity of the autoviral population. 454 is a simple, efficient, and cost-effective means of obtaining approximate genetic diversity in a sample. In exemplary embodiments, DNA vectors and plasma RNA are amplified with barcoded primers and then sequenced using 454 JRs to obtain an average of approximately 2000 reads per amplicon / sample.

[0164] The present invention will be described in more detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to limit the invention unless otherwise specified. Accordingly, the present invention should not be construed as being limited to the following examples, but rather as encompassing all variations that become apparent as a result of the teachings provided herein. [Examples]

[0165] This example presents immune reconstitution and virological outcomes from two independent clinical trials in which HIV-infected adults received a single infusion of CCR5 gene-edited CD4+ T cells. In the first study (clinical trial SB-728-0902), this intervention was found to increase CD4+ T cell counts and restore global T cell homeostasis in a group of individuals who had previously failed to normalize their CD4+ T cell counts despite long-term effective ART. Importantly, a significant long-term decay in total HIV reservoir size was observed in the majority of participants, with 1 log per million cells in 4 out of 9 participants. 10A decrease in HIV DNA exceeding copies was observed. These results were generated using HIV DNA measurements that did not include HIV replication-eligible reservoirs, but the very significant observed decrease in HIV reservoirs is in stark contrast to the very stable levels reported in recent clinical trials with long-term ART and latent infection reactivators. These outcomes are indicative of non-infected CD45RA. int RO int T SCM This was thought to be due to the serial replacement of short-lived HIV-infected cells with derived cells. In the second study (SB-728-1101 clinical trial), a single injection of these CCR5 gene-edited cells resulted in this novel CD45RA int RO int T SCM We confirmed that the generation of subsets and the increased frequency of such cells were associated with improved control of HIV replication after ART discontinuation. These observations support a model in which CCR5 gene editing of memory CD4+ stem cells allows these cells to proliferate and differentiate into other memory subsets in the presence of the virus, protecting their offspring from infection.

[0166] material and method The SB-728-0902 clinical trial was a phase 1, non-comparative, open-label, non-randomized study in chronically HIV-infected patients treated with ART (ClinicalTrials.gov#NCT01044654). This study was sponsored by Sangamo Therapeutics and conducted at two centers in the United States between December 2009 and April 2014. The primary objective of the study was to evaluate the safety and tolerability of escalating doses of autologous CD4+ enriched T cells (SB-728-T cells) edited in the CCR5 gene by ZFN. Secondary objectives included evaluating the increase in CD4+ T cell count, the long-term persistence of CCR5 gene-edited cells, homing to the intestinal mucosa, and the impact on HIV viral persistence (HIV RNA and proviral DNA). A total of nine participants were enrolled in three escalating dose cohorts (three participants in each cohort). All participants were followed weekly for the first four weeks, then monthly for one year, and subsequently enrolled in a three-year safety trial. Participant 1-01 experienced treatment discontinuation between months 12 and 31.

[0167] The SB-728-1101 clinical trial was a phase 1, non-comparative, open-label, non-randomized study in chronic HIV-infected patients treated with ART (ClinicalTrials.gov#NCT01543152). This study was sponsored by Sangamo Therapeutics and conducted at 12 centers in the United States between March 2012 and January 2017. The primary objective of this study was to evaluate the safety and tolerability of escalating doses of cyclophosphamide (CTX) pretreatment to promote CD4+ T cell proliferation after a single dose of SB-728-T cells. Participants received 0.1 (Cohort 1, n=3), 0.5 (Cohort 2, n=6), 1.0 (Cohort 3, n=3), 1.5 (Cohort 5, n=3), and 2.0 g / m² the day before SB-728-T cell infusion. 2Cohort 4 (n=3) received CTX at a dose of [specified dose]. Subsequently, participants received approximately 10 to 40 billion SB-728-T cells. All participants were followed weekly for the first four weeks, every other month until week 14, monthly until week 22, and then every two months until month 12. ART was discontinued for 16 weeks, 6 weeks after SB-728-T infusion (Figure 17A). A secondary objective was to evaluate the effect of SB-728-T cells on plasma HIV-1 RNA levels after ART discontinuation. ART was resumed in participants whose CD4+ T cell count decreased to less than 500 cells / μL during treatment discontinuation, and / or whose HIV-RNA increased to more than 100,000 copies / mL in three consecutive weekly measurements. With the exception of one participant who dropped out of the study, all participants completed the one-year study and were enrolled in a three-year long-term safety study. One participant (03-003) did not discontinue ART.

[0168] The final clinical protocols, amendments, and informed consent documents were reviewed and approved by the NIH Recombinant DNA Advisory Board and the institutional review boards and biosafety boards of each research center (as required). All participants provided written informed consent.

[0169] Registration Criteria SB-728-0902 Test Eligible participants were 18 years of age or older and infected with HIV as documented by ELISA. Participants were aviremia (undetectable HIV RNA), receiving stable ART with CD4+ T cell counts of 200–500 cells / μL, had adequate intravenous access, and had no contraindications to leukocytosis. Key exclusion criteria included SNPs in the CCR5 zinc finger nuclease target region, current or previous AIDS diagnosis, treatment with maraviroc or immunosuppressants, and co-infection with hepatitis B or C.

[0170] SB-728-1101 Test Eligible participants were 18 years of age or older and infected with HIV as documented by ELISA. Participants were aviremia to stable ART with CD4+ T cell counts >500 / μL, infected with R5-tropic HIV, and voluntarily discontinued their current ART during treatment interruption. Key exclusion criteria included more than 40 adenovirus neutralizing antibodies, SNPs in the CCR5 zinc finger nuclease target region, current or previous AIDS diagnosis, treatment with maraviroc or immunosuppressants, and co-infection with hepatitis B or C.

[0171] Cell manufacturing In short, participants underwent 10L of leukocyte apheresis to collect, enrich, modify, and proliferate autologous CD4+ T cells. SB-728-T refers to autologous CD4+ enriched T cells transduced ex vivo with SB-728, a replication-deficient recombinant Ad5 / 35 viral vector encoding CCR5-specific ZFNs (SBS8196z and SBS8267), and includes a mixture of gene-edited and unedited cells. Expression of CCR5-specific ZFNs induces double-strand breaks in the cell's DNA, which are repaired by cellular mechanisms, leading to random sequence insertions or deletions (indels) in approximately 25% of transduced cells. These indels disrupt the CCR5 coding sequence, leading to frameshift mutations and termination of protein expression.

[0172] Cryopreserved peripheral blood mononuclear cell (PBMC) samples SB-728-0902 The availability of cryopreserved samples at different time points varied among participants, and as a result, time points were grouped into early (14–28 days post-infusion), mid-term (4–7 months or 9–10 months), late (11–12 months), and long-term (2–3 or 3–4 years) post-infusion time points. Baseline samples included cryopreserved PBMCs from the first leukocyte apheresis (2–3 months prior to infusion) and a small blood draw 1–2 weeks prior to infusion. PBMCs from participants 1-01, 1-02, and 1-03 were not cryopreserved until 6 or 8 months post-infusion. Most participants agreed to undergo large blood draws (n=9, 2–3 years) and / or leukocyte apheresis (n=7, 3–4 years) during the long-term follow-up period to enable assays requiring large volumes of cells, such as CCR5 sequencing and integrated HIV DNA quantification in selected CD4+ T cell subsets. For certain assays, including the ICS assay and CD95 flow cytometry staining, baseline samples were still available for only six participants. Manufacturing samples (SB-728-T product) were also available to all participants.

[0173] SB-728-1101 Clinical measurements (CD4, CD8 count, viral load (VL), and pentamer replication markers) were performed at all time points. Availability of cryopreserved PBMCs at baseline and pre-ATI was unavailable for participants in cohorts 1 and 2; consequently, immunological (T cell phenotyping, CCR5 DNA sequencing for ZFN-mediated mutations in selected CD4+ subsets) and virological (integrated HIV DNA) measurements were performed only in participants in cohorts 3–5. Baseline samples included cryopreserved PBMCs from initial leukocyte apheresis (2–3 months prior to infusion) and small blood draws (1–2 weeks prior to infusion). Manufactured samples (SB-728-T product) were also available to participants in cohorts 3–5.

[0174] Rectal and lymph node biopsy Rectal biopsies were performed on participants in the SB-928-0902 trial at baseline, day 14, 3, 6, and 12 months (n varied between 3 and 9 participants per time point). Mucosal mononuclear cells were isolated from sigmoid colon biopsies obtained by endoscopy via a combination of collagenase digestion and 18G needle teasing. Inguinal lymph nodes were biopsied from three volunteers at a time point (9–18 months after SB-728-T infusion). Anton et al. 47 As described, the tissue was processed into single cells, and genomic DNA was isolated for evaluation of CCR5 gene modifications.

[0175] Quantification of CCR5 gene-edited CD4± T cells by polymerase chain reaction ZFN-mediated gene modification can generate a wide range of frameshift mutations by disrupting the CCR5 locus. A PCR-based assay was developed to measure the acquisition of intrinsic replication of the 5-nucleotide (pentamer) DNA sequence, CTGAT, at approximately 25% of ZFN cleavage sites of gene-edited alleles. Genomic DNA (gDNA) was extracted from PBMCs using a commercially available kit (Masterpure DNA Purification Kit, Epicenter, Madison, WI). Standard PCR was performed using 5 μg of gDNA to amplify a 1.1 kb region containing CCR5 gene modification. This 1.1 kb amplicon was then evaluated by two independent qPCRs: one specific to the pentamer-replication-CCR5 gene-edited allele (by using primers containing pentamer replication), and the second amplifying all CCR5 alleles. The ratio of pentamer-replication-specific templates and the total number of CCR5 alleles give pentamer replication per 1 million PBMCs. The assay was performed 10 5 Each total number of CCR5 alleles has sensitivity to one CCR5 gene-editing allele. The frequency of CCR5 gene-edited cells in PBMCs was estimated by multiplying the frequency of pentameric replicating gene-edited cells by 4.

[0176] Quantification of CCR5 gene modification in SB-728-T product using Cel-I Cel-I nuclease specifically cleaves DNA double strands at strain sites created by either bulges or mismatches in the double-helix DNA structure. A protocol using this enzyme was employed for the quantification of minor indels typically induced by ZFN-mediated gene modification. Briefly, the genomic region of interest (CCR5) is PCR-amplified, the PCR product is denatured, and then re-annealed to allow the wild-type and non-homologous end-joint editing alleles to anneal together to create a heteroduplex. The re-annealed PCR product is then digested with Cel-I nuclease, cleaving the PCR-amplified DNA at mismatch sites. The level of ZFN-mediated gene modification can then be quantified by determining the ratio of the uncleaved parental fragment to the two lower-mobility cleavage products.

[0177] Quantification of CCR5 gene modifications by next-generation sequencing / MiSeq The locus of interest (ZFN binding site of CCR5) was PCR-amplified from genomic DNA, and the modification level at each locus was determined by paired-end deep sequencing on an Illumina MiSeq sequencer. Paired sequences were merged via SeqPrep (John St. John, https: / / github.com / jstjohn / SeqPrep, unpublished). Needleman-Bunsch alignment was performed between the target amplicon genomic region and the resulting Illumina sequence to map indels. CCR5 sequencing was performed on selected CD4+ T cell subsets from SB-728-0902 participants using SB-728-T product (n=9) and samples from 3-4 years (n=8), as well as from SB-728-1101 cohort 3-5 participants using SB-728-T product (n=7) and samples from 6 and 22 weeks (n=7). The number of CCR5 gene-edited memory subset cells was estimated by multiplying the number of memory subset cells by the frequency of the CCR5 gene-edited allele within each memory subset, which is determined by CCR5 sequencing.

[0178] CCR5 gene editing CD45RA after SB-728-T injection int RO int T SCM Cell tracking Using sequencing of CCR5 ZFN-mediated mutations in selected CD4+ T cell subsets, post-injection CD45RA int RO int T SCM Cell differentiation was tracked. First, sequences detected in wild-type CCR5 (amplicon) and in only one of the sequenced samples were excluded from further analysis (approximately 80% of unique CCR5 sequences). Then, for each donor, the CD45RA of the SB-728-T product was analyzed. int RO int T SCMSequences expressed only in cells were identified, and their distribution in CD4+ T cell memory subsets in samples from 3-4 years old (SB-728-0902) or samples from 6 and 22 weeks old (SB-728-1101) was then analyzed.

[0179] Estimated expansion of SB-728-T after injection The level of the CCR5 gene-edited allele surviving in participants relative to the amount of injected CCR5 gene-edited cells can be estimated using measurements of CCR5 modification by pentameric replication markers and assumed CD4+ cell counts. 1) Blood volume is 4.7 liters, 2) Approximately 2.5% of all CD4+ T cells are peripheral. 49 The distribution of the SB-728-T product observed in these cells, as well as in endogenous CD4+ T cells (the level of CCR5 modification in CD4+ T cells from sigmoid and inguinal nodules is similar to that in the periphery, Figure 8E).

number

[0180] Cellular phenotyping of SB-728-T products in samples at baseline and after SB-728-T injection. Analysis and evaluation of co-inhibitory receptors on CD4+ and CD8+ subsets were performed before fixation with 2% FA (Sigma Aldrich) at 22°C for 15 minutes. SCM The procedure was performed using 1 million thawed PBMC surfaces stained at 4°C for 30 minutes with either a panel or a negative regulatory factor panel. Both panels included CD3 Alexa 700 (clone UCHT1) (BD Biosciences), CD4 Qdot 605 (clone S3.5) (Invitrogen), CD27 APCe780 (clone O323) (eBioscience), CD8 PerCP (clone SK1), CD45RA BV650 (clone HI100), CD45RO PerCPe710 (clone UCHL1) (Biolegend), and aqueous fluorescently reactive dye (dead cell marker) (Invitrogen).SCM The panel includes CD95 PE-Cy7 (clone DX2), CD58 PE (clone 1C3), CD127 BV421 (clone HIL-7R-M21), CD28 APC (clone CD28.2), CD14 V500 (clone M5E2) (BD Biosciences), CD19 BV510 (clone H1B19) (Biolegend), and CCR7 FITC (clone 150503) (R&D). The negative regulatory panel included CCR7 PE-CF594 (clone 150503), CTLA-4 APC (clone BNI3), CD31 PE (clone WM59) (BD Biosciences), Tim-3 BV421 (clone F38-2E2), PD-1 PE-Cy7 (clone EH12.2H7) (Biolegend), and LAG-3 FITC (clone 17B4) (Novus Biologicals). A minimum of 100,000 live cells were obtained within 24 hours using BD LSR-II and analyzed using FlowJo version 9.

[0181] Cell sorting For the quantification of pentameric replication makers and integrated DNA levels within CD4+ T cell subsets, CD4+ T cells were first isolated from PBMCs by negative magnetic selection (StemCell), followed by CD3 Alexa 700 (clone UCHT1), CD95 PE-Cy7 (clone DX2), CD58 PE (clone 1C3), CD127 BV421 (clone HIL-7R-M21), CD28 APC (clone CD28.2), CD14 V500 (clone M5E2) (all from BD Biosciences), CD4 Qdot 605 (clone S3.5) (Invitrogen), CD27 APCe780 (clone O323) (eBioscience), CD8 PerCP (clone SK1), CD45RA BV 650 (clone HI100), CD45RO PerCPe710 (clone UCHL1), and CD19 BV Cells were surface-stained with 510 (clone H1B19) (Biolegend), CCR7 FITC (clone 150503) (R&D), and aqueous fluorescent dye (Invitrogen). Up to 200,000 total CD4+ T cells and CD4+ T cell subsets were then sorted using FACSAria (Becton Dickinson) and stored as dry pellets at -80°C until analysis. For gene array analysis of immunosubsets, 10,000 sorted cells were collected directly into 1.5 mL Eppendorf tubes without RNAse containing 500 μL of RLT buffer with 1% β-mercaptoethanol and stored at -80°C until analysis.

[0182] HIV DNA in PBMCs, total and selected CD4± T cell subsets Total HIV DNA in PBMCs was measured by droplet digital polymerase chain reaction. In short, genomic DNA (gDNA) was extracted from PBMCs using a commercially available kit (Masterpure DNA Purification Kit, Epicenter, Madison, WI). 2 μg of gDNA was digested with restriction enzyme DdeI at 37°C for 1 hour. PCR droplets were prepared according to the manufacturer's recommendations. Briefly, 20 μL of multiplex PCR mixture was prepared by mixing 250 ng or 500 ng of digested gDNA with ddPCR® 2× master mix and two Taqman primer / probe sets. PCR droplets were generated in a DG8® cartridge using a QX-100 droplet generator, and each 20 μL of PCR mixture was divided into droplets of approximately 15,000 nanoliters in size. The PCR droplets were transferred to a 96-well PCR plate and sealed with foil. Standard PCR was performed using a Bio-Rad C1000 thermal cycler (40 cycles of 95°C (60 sec), 94°C (30 sec) / 60°C (60 sec), 98°C (600 sec)). HIV DNA copy number was assessed using a QX-100 droplet digital PCR system (Bio-Rad, Hercules, CA). PCR-positive and PCR-negative droplets for HIV gag and RPP30 were measured, and template concentrations were calculated by Poisson analysis. HIV copy number was determined by normalizing the HIV gag concentration to the RPP30 concentration. Integrated DNA was measured as described above in purified CD4+ T cells from baseline SB-728-0902 participants, SB-728-T product, 2-3 year samples (n=9), and SB-728-T product and selected CD4+ T cell subsets from 3-4 year samples (n=8). Integrated DNA was measured in purified CD4+ T cells, as well as in selected CD4+ T cell subsets from SB-728-1101 cohort 3-5 participants in baseline, 2-6 weeks, and 14-22 weeks samples (n=8).

[0183] HIV tropism assay HIV tropism was assessed using the commercially available Trofile® DNA assay (Monogram BioSciences / LabCorp, South San Francisco, CA). Viral envelope DNA sequences were extracted from PBMCs. HIV tropism is determined using a cell-based transduction assay in which HIV env protein sequences are amplified from PBMC samples, subcloned as a library, packaged in a lentiviral vector, and evaluated using co-receptor restriction cell lines.

[0184] Intracellular cytokine staining After thawing the PBMCs and allowing them to stand for 12 hours, 2 million cells each were stimulated for 6 hours with either brefeldin A (5 μg / mL) (Sigma Aldrich) and gag peptide (1 μg / peptide / mL, NIH AIDS reagent program), Staphylococcus enterotoxin B (SEB, 1 μg / mL), or complete medium (mock). Next, the cells were surface-stained with CD3 Alexa 700 (clone UCHT1), CD8 Pacific Blue (clone RPA-T8), CCR7 PE-CF594 (clone 150503), CD14 V500 (clone M5E2) (BD Biosciences), CD4 Qdot 605 (clone S3.5), CD27 APCe780 (clone 0323) (Invitrogen), CD45RA BV 650 (clone HI100), CD19 BV 510 (clone H1B19) (Biolegend), and aqueous fluorescent dye (Invitrogen), then permeabilized with 0.05% saponin, and stained with IL-2 PerCP-Cy5.5 (clone MQ1-17H12), IFNγ APC (clone B27), and TNFα Alexa Fluor 488 (clone MAB11) (BD After intracellular staining with Biosciences, cells were fixed with 2% formaldehyde. Cells were acquired within 24 hours using BD LSR-II. At least 500,000 bioevents were acquired. Cells were analyzed using FlowJo version 9, and the distribution of the multifunctional CD8+ T subset was determined using a Boolean gating function.

[0185] T cell receptor (TCR) repertoire TCR repertoire analysis was performed using the immunoSEQ assay (Adaptive Biotechnologies, Seattle, WA). The immunoSEQ method amplifies TCR CDR3 sequences rearranged by multiplex PCR to search for all Vβ and Jβ combinations from isolated genomic DNA, and then sequences the TCR CDR3 strands using high-throughput sequencing techniques to determine the composition of various T cell clones within each sample. TCR diversity is assessed using the Shannon entropy index, which explains both the number of unique clones of TCR Vβ CDR3 sequences present in each sample (abundance) and the distribution of clones (uniformity). A larger Shannon entropy index reflects a more diverse distribution of TCR Vβ CDR3 sequences.

[0186] Gene microarrays and analysis The selected CD4+ or CD8+ subset was sorted into the RLT buffer as described above. Specifically, CD4+ T CM CD4+ T TM CD4+ T EM CD8+ total memory cells were selected at baseline and 12 months. In addition, the CD4+ memory subset was also selected at 3-4 years and CD45RA int RO int T SCM , T CM , and T EMThe samples contained cells. Selected cells were lysed for RNA extraction according to the manufacturer's instructions (Qiagen, Valencia, CA). In vitro transcription was performed following a T7 oligo(dT) prime reverse transcription reaction. These products underwent a second amplification (MessageAmpII aRNA amplification kit by Life Technologies) to generate biotin-labeled aRNA, which was hybridized to an Illumina Human HT-12 version 4 Expression BeadChip according to the manufacturer's instructions and quantified using the Illumina iScan system.

[0187] Analysis of gene array output data was performed using the R statistical language and the Linear Model for Microarray Data (LIMMA) statistical package from Bioconductor. Briefly, scanned array images were inspected for artifacts and anomalous signal distributions within the chip, and arrays with low overall intensity or variability were excluded from the analysis. Inter-chip hybridization quality was assessed using diagnostic plots such as density plots, box plots, and heatmaps of inter-array distances. Intensities were log2 transformed before normalization using quantile normalization. Probes and control probes that did not map to annotated RefSeq genes were excluded. Differentially expressed gene analysis was performed at 12 months compared to baseline for CD4+ T CM (n=6), CD4+ T TM (n=9), CD4+ T EM The procedure was performed on (n=7) and CD8+ total memory cells (n=9), and CD4+ T cells were observed in 3-4 years. CM and T EM Compared to the subset, CD4+ CD45RA int RO int T SCMThe above study was conducted (n=7). Differences in gene expression levels between different time points or subsets were determined by performing longitudinal donor pair analysis. The statistical significance (P<0.05) of differential gene expression between baseline and month was assessed using adjusted t-tests implemented in the LIMMA package. All microarray data are deposited with GEO under accession number GSE66214.

[0188] We used gene set enrichment analysis (GSEA) to identify enriched biological pathways regulated in T memory cells after injection (Figure 4). GSEA is a statistical method that determines whether members of a particular gene set preferentially occur higher or lower on a ranked gene list, where genes are ranked according to their strength of association with outcomes of interest. More specifically, GSEA calculates an enrichment score (NES) that reflects the extent to which a gene set accounts for a large proportion among genes with different levels of expression. The significance of the observed NES is obtained by a sorting test, and the gene list is re-sorted to determine how often the observed NES occurs by chance. We then performed a state-of-the-art analysis to examine specific genes in the gene sets that contribute most to enrichment. Using the GSEA pre-ranked gene list option, we tested enrichment of curated and custom gene sets from MSigDB (http: / / software.broadinstitute.org / gsea / msigdb / ) to test enrichment of Treg and STAT3 pathways in our data. Gene sets with a false discovery rate (FDR) exceeding 25% and a nominal p-value exceeding 0.05 were discarded.

[0189] Using the GSEA described above in conjunction with the Fischer combined testing approach, CD4+ CD45RA int RO int T SCM vs CD4+ T CM Comparison of CD4+ and CD45RA int RO int T SCM vs CD4+ T EMIn the comparison, we identified the pathways that were enriched. EM and T CM Compared to both, CD45RA int RO int T SCM Selected pathways that were significantly enriched in the genes induced or repressed within the study were grouped into several biological functions: cell cycle, cellular metabolism, cytokine signaling, Notch signaling, and apoptosis (Figure 4A).

[0190] Using a pie chart, CD4+ T CM , T EM and T TM Furthermore, in the total CD8+ memory subset, the top enriched pathways that increased or decreased at 12 months compared to baseline were represented (Figure 4B, C). To assess whether the gene expression profiles of the CD4+ memory T cell subset at 12 months post-infusion mimicked the profiles of immune responders (IRs), GSEA analyses were performed in cohorts of HIV-infected IRs (n=20, CD4+ count >500 cells / μL) and immunological non-responders (INRs, n=21, CD4+ count <350 cells / μL) from an independent cohort (Cleveland Immunodeficiency-CLIF-Cohort). All pathways (5 / 5) upregulated in CD4+ memory T cells at 12 months post-SB-728-T product infusion were richer in IRs compared to INRs (Figure 4E, F), and these pathways were associated with active metabolism (MYC, OX / PHOS) and proliferation (DNA repair).

[0191] CD4+ T at 12 months compared to baseline CMTo investigate the effects of the SB-728-T product on inflammation and immune activation in total CD8+ memory cells, we first performed longitudinal donor pair analysis using the LIMMA approach as described above. Genes were considered differentially expressed between baseline and 12 months if the probability P was less than 0.05. Differentially expressed genes induced by interferon type I (IFN I) were identified using the interferon database (http: / / interferome.its.monash.edu.au / interferome / home.jspx). Selected genes (upregulated or downregulated at 12 months compared to baseline) were chosen from each comparison and sent to the GeneMania web server (http: / / www.genemania.org / ) to generate gene interaction networks using co-expression interaction categories.

[0192] Using linear regression analysis, we analyzed the 10 data from years 2 to 4. 6 Frequency of total HIV DNA copies per individual PBMC and CD45RA at 3-4 years int RO int T SCM CD45RA in samples from 3-4 years old correlated with the number of CD45RA int RO int T SCM The gene expressed by CD45RA was identified. int RO int T SCM A linear model was fitted (using the R language) between gene expression and the levels of these outcomes as continuous variables, and GSEA was used to positively or negatively correlate pathways with both readouts. Pathways regulated in a similar manner and pathways regulated in different directions are shown in Figure 4g. Measured by total HIV DNA at years 2–4, HIV reservoir size was positively correlated with CD45RA at years 3–4. int RO int T SCMTop pathways negatively correlated with counts were highlighted by plotting normalized enrichment scores (Figure 4H, I). This analysis was performed on 6 of the 7 participants. Post-injection CD45RA int RO int T SCM Participant 1-02, who had a low cell engraftment rate, was identified as an outlier in the exploratory analysis and was therefore excluded from the analysis.

[0193] statistical analysis HIV reservoir analysis The overall decay of the HIV reservoir after infusion over time (number of days) (10 6 The model was constructed using a mixed-effects linear regression model with random intercepts (measured as total HIV DNA per PBMC), as implemented in the function lmer from the R package lme4. The p-values ​​associated with the model were estimated using the cftest function, as implemented in the multicomp package in R.

[0194] To analyze the decay of the HIV reservoir in each individual, we used 10 data points over time. 6 The frequency of total HIV DNA per PBMC was fitted to each individual using a linear regression model with GraphPad Prism v7.0 software. P-values ​​and regression coefficients were calculated (Figure 2A). Missing baseline values ​​for participants 1-01 and 1-02 were calculated using model-fitted intercepts.

[0195] Clinical data, CCR5 modification, and flow cytometry Nonparametric donor-paired two-sided analyses were performed using a paired Wilcoxon rank-sum two-sided test to compare changes in total CD4+ T cell and subset numbers, CD4:CD8 ratio, T cell function, immune checkpoint inhibitors, and integrated HIV DNA after injection, compared to baseline. The Wilcoxon rank-sum two-sided test was also used to compare levels of CCR5 gene-editing alleles between subsets, and to compare TCR repertoire and CCR5 gene-editing allele diversity between the SB-738-T product and long-term time points. In the SB-728-0902 study (pentamer replication and CCR5 DNA sequencing), unpaired nonparametric two-sided comparisons were performed using the Mann-Whitney two-sided test for, for example, the frequency of CD95+ cells after injection compared to baseline, and levels of CCR5 gene-editing alleles between different CD4+ memory subsets, where the number of matched participants varied by time point and fewer than six matched pairs were included at a given time point. Nonparametric correlation analyses were performed between various measures and clinical outcomes, including delta-CD4+ T cell count (SB-728-0902), reservoir size change calculated using the ratio of the last measured value to baseline (at years 2–4) (SB-728-0902), and control of viral replication (SB-728-1101), using Spearman's rho(ρ) test. Multiple comparison tests were controlled by calculating FDR values ​​using the original FDR method of Benjamini and Hochberg. P-values ​​< 0.05 and Q-values ​​< 0.25 were considered significant. These statistical analyses were performed using GraphPad Prism v7.0.

[0196] Statistical analysis of total HIV DNA decay after injection of SB-728-T product in SB-728-0902 Statistical analysis was performed using Monolix version 2016R1 (http: / / lixoft.com / products / monolix / ), a statistical software package developed by Marc Lavielle and implemented in Matlab, to estimate parameters using a nonlinear mixed-effects model approach to explain the post-injection decay of HIV DNA observed in six participants. Biphasic exponential decay was estimated using the equation Y = a + b1*exp(-r1*time) + b2*exp(-r2*time) (Equation 1) using both individual and group approaches (combining all six patients). Monolix implements a stochastic approximation of the expectation maximization algorithm using a Markov chain Monte Carlo (MCMC) iterative algorithm. The MCMC iteration method uses the Metropolis-Hastings approach. Different distributions were considered for each of the biphasic decay model equations to perform the fitting. The two decay rates r1 and r2 took values ​​between (0,1) and were estimated according to a logit-normal distribution. The slow and fast intercept parameters b1 and b2, along with the plateau parameter a, take only positive values ​​and were estimated using a log-normal distribution. To ensure model convergence, 2000 Monte Carlo iterations were used in the simulation step. The shape of each individual fit of Monolix was also verified for the same two-phase decay function using the nonlinear least-squares estimation method, the nlsLM package in R.

[0197] To estimate the time at which the fast and slow phases of HIV DNA began to dominate, the following equations were used to plot the gradients associated with the fast and slow phases, respectively. Gradient_Fast = (a+b1+b2)*exp(-r1*time) (Equation 2) Gradient_Slow=(a+b2)*exp(-r2*time)(Equation 3)

[0198] The intersection of the two gradients (equations 2 and 3) represents the time when the slow phase begins to dominate the fast decay phase. Similarly, the intersection of the slow phase (equation 3) and the plateau line indicates the time when the slow decay phase ends and HIV DNA reaches the new frequency level estimated by the plateau. Each calculation for the six patients was performed using MATLAB.2016.

[0199] Similarly, the following equations were used to estimate the contributions of both the fast and slow phases to total HIV DNA decay. Percentage_Fast: Φ = b1 / (Y0 - plateau) * 0.01, Y0 = a + b1 + b2 (HIV DNA copy at t=0 days) Percentage_low speed: Y=100-Φ

[0200] Model selection and diagnosis Compared to the results of the collective approach, the individual approaches yielded better fits. The Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) were used to evaluate the differences between R-fits and Monolix-fits, as well as the individual-versus-population fitting results. Smaller AIC and BIC scores indicate the most parsimony, which was better achieved using Monolix's individual approaches.

[0201] A mathematical model of the dynamics of CD4± T cells To investigate the persistence of CCR5 gene-edited memory CD4+ T cells, we developed a mathematical model to explain the dynamics of the memory CD4+ T cell population. Memory CD4+ T cells were divided into two populations: CCR5 gene-edited and non-edited memory CD4+ T cells. The model included naive (N) and stem memory CD45RA. int RO int (TSCM2, TSCM2 GE ), Main memory CD45RA + (TSCM1, TSCM1 GE ), Central Memory (CM, CM) GE ), Transitional Memory (TM, TM) GE) and effector memory (EM, EM GE Considering this, the subscript GE refers to CCR5 gene-edited CD4 T cells. The differential equation describing this system is as follows:

number

[0202] Using the individual fitting approach of Monolix R2018, we fitted the ODE model with both CCR5 gene-edited and non-gene-edited CD4+ T cell memory subset data from five patients with extended ATI interruption periods (weeks 6–month). Due to the small sample size, individual fitting routines were employed to parameterize the model parameters, as described in the previous section (statistical analysis of total HIV DNA decay after SB-728-T product injection in SB-728-0902). A log-normal distribution with only positive values ​​was assumed for all model parameters.

[0203] Global Sensitivity Analysis To determine which parameters most significantly influence cell population size, sensitivity analysis tests were performed using Latin hypersquare sampling (LHS) and partial rank correlation coefficients (PRCC) to measure the linear relationship between model parameters and model outputs. This test allows for simultaneous examination of the sensitivity between multiple parameters and model outputs. To account for the uncertainty in the parameter distribution, a uniform distribution was used to vary the model parameters, where the maximum and minimum values ​​were taken from five individual fits obtained for each subject. A monotonic relationship between model parameters and outputs was confirmed. To ensure accuracy, PRCC values ​​were obtained using 100,000 bins in MATLAB.

[0204] Estimation of HIV DNA decay due to dilution of injected cells in SB-728-0902 Post-injection HIV DNA decay in participants of the SB-728-0902 study, due to dilution by the volume of injected cells, can be estimated using CCR5 gene editing with pentamer replication markers and Cel-I nuclease measurements in the SB-728-T product, assuming that 1) one gene-edited allele represents one gene-edited cell, 2) CD4+ T cells from the SB-728-T product do not contain cells with HIV DNA, and 3) unedited cells persist as well as CCR5 gene-edited cells after injection (participants continued ART).

[0205] Estimated frequency of CCR5 gene-edited cells in PBMCs = 10 at each time point 6 Frequency of pentamer replication per PBMC * 4 / 1000

[0206] Estimated frequency of injected cells in PBMCs = Frequency of CCR5 gene-edited cells in PBMCs at each time point * (100 / Frequency of CCR5 gene-edited cells in SB-728-T product, determined by Cel-I nuclease)

[0207] Estimated decay of HIV DNA due to injected cells = Baseline frequency of cells containing HIV DNA * Frequency of injected cells in PBMCs at each time point / 100

[0208] Estimated frequency of HIV DNA from injected cells = Baseline frequency of cells containing HIV DNA - Estimated decay of HIV DNA by injected cells at each time point

[0209] outliers Participants 1-02 had elevated anti-adenovirus titers and exhibited engraftment levels in CCR5 gene-edited cells and a highly enriched population of CD45RA within the gene-edited cells. int RO int T SCMCell persistence may be impaired (SB-728-T product is derived from transduction with a recombinant Ad5 / F35 adenovirus vector encoding a CCR5-targeted ZFN), therefore, CCR5 gene-edited cells and CD45RA int RO int T SCM Selective analyses focusing on correlating subset proliferation with HIV reservoir decay were excluded (e.g., Figures 1c-d, 2d-e, and 3g-i).

[0210] result A single SB-728-T injection resulted in a continuous reduction in HIV reservoir size, which correlates with the proliferation and persistence of CCR5 gene-edited cells. Clinical trial SB-728-0902 evaluated nine HIV-infected adults undergoing long-term ART who were unable to increase their CD4+ T cell count to above 500 cells / μL. At baseline, participants had received effective ART for 7–22 years and had a mean CD4+ T cell count of 363 cells / μL. CD4+ T cell count was inversely correlated with the level of integrated HIV DNA (referred to here as HIV reservoir) at baseline visit (P=0.017). All participants received a single infusion of ZFN-mediated CCR5 gene-edited CD4+ T cells.

[0211] Peripheral CD4+ T cell counts (and CD4:CD8 ratio) increased within 7 days post-infusion, as expected (see online discussion, Figures 10A, 10B). Notably, this increase persisted, with CD4+ T cell counts remaining significantly above baseline for 3–4 years during longitudinal observation (P=0.024) (Figure 10A). Proliferation of CCR5 gene-edited cells peaked 7–21 days post-infusion (median 2.4-fold proliferation at 21 days, Figure 10c) and was associated with the increase in CD4+ T cell counts. CCR5 gene-edited CD4+ T cells were detected in PBMCs for up to 4 years (mean 0.8% marked PBMCs and mean 2.7% marked CD4+ T cells) and in rectal biopsies and lymph nodes for up to 12 months (at the last measured time) (Figures 10d, 10e).

[0212] Next, we investigated whether the recovery of CD4+ T cell counts after SB-728-T injection led to a reduction in the frequency of circulating cells containing HIV DNA. Compared to baseline, a significant decrease in total HIV DNA levels was observed at 2 years post-injection (P=0.0195, mean decay -0.91 log10, 95% confidence interval (CI) -1.71 to -0.11) (Figure 2A). Furthermore, a significant decay in the frequency of CD4+ T cells with integrated DNA (Figure 2B) was also observed after injection. Higher levels of CCR5 gene-edited cell proliferation at early (21 days) and long-term (approximately 3-4 years) time points correlated with a greater sustained reduction in HIV DNA levels (r 2 =0.62, P=0.0014 and r 2 =0.91, 0.0003 respectively) (Figures 2C and 2D). These results strongly suggest that the persistence of injected CD4+ T cells influences the decay of HIV reservoir size.

[0213] Interestingly, CD4+ T cells from the SB-728-T product had a significantly lower frequency of latent infected cells than CD4+ T cells from baseline (P=0.004, Figure 16A). Using a biphase decay model, we determined whether the persistence of injected cells containing low levels of integrated HIV DNA contributed solely to the decay of the HIV reservoir through dilution during the peak proliferation of injected cells. The gradient of HIV DNA decay was maximum during the first 1–15 days of injection, during which a mean decay of 30.47% (95% CI, 9.664–51.28) was observed. Subsequently, HIV DNA levels continued to decline more slowly with a half-life of 211 days (95% CI, 56–365). This slower decay phase accounted for the majority of the HIV DNA decline (mean 69.5% of the decline) (Figure 2E). Subsequently, the estimated frequency of cells containing HIV DNA as a result of dilution was calculated at each time point (Figure 2F). The observed frequency of cells containing HIV DNA was found to be lower after approximately 100 days than the frequency estimated by dilution alone, demonstrating that dilution alone cannot explain the long-term decrease in the frequency of HIV-infected cells. Our analysis suggests that the persistence of injected cells leads to HIV decay through mechanisms that may include the restoration of T cell homeostasis and / or replenishment of the CD4+ T cell pool by uninfected cells.

[0214] A novel memory stem cell-like CD4± T cell subset contributes to the restoration of T cell homeostasis and correlates with reservoir decay. To investigate the mechanisms leading to CD4+ T cell rearrangement, we performed a longitudinal analysis of the distribution of CD4+ T cell subsets after injection. Our study focused on intermediate levels of CD45RA and CD45RO ("CD45RA"). int RO int It showed a specific increase in T cells expressing CD45RA (Figure 3A). int RO int Cells present in the SB-728-T product were highly enriched in the CCR5 gene-edited allele, and their absolute numbers increased significantly at all time points analyzed after injection. Importantly, CD45RA after injection int RO intChanges in cell number were significantly correlated with long-term increases in CD4+ T cell count, rather than with changes in other memory subsets (Table 1). CD95+CD58+ cells (CD45RA int RO int and CD45RA + RO - T within a subset SCM The frequency of the marker expressed within the cells was positively correlated with the proliferation of CCR5 gene-edited cells. The level of the pentamer replication marker (sequence tag of CCR5 gene-edited cells) was positively correlated with CD45RA int RO int CD95+ cells (CD45RA int RO int T SCM (and referred to as) and CD45RA + RO - CD95+ cells (CD45RA + T SCM They are specifically concentrated within (what is called) the central memory (T) of the 3rd to 4th year. CM ) or transitional memory (T TM Compared to ) cells, CD45RA int RO int T SCM The levels were approximately 14 and 21 times higher than the intermediate levels (Figure 3B). Sequencing of CCR5 DNA mutations driven by gene editing was performed at T CM 2.5% to 16.7% in T TM 1% to 16.7% in T EM Compared to the range of 0.7% to 2.59% in CD45RA, int RO int T SCM Long-term enrichment of the CCR5 gene editing allele was confirmed in the range of 14.4% to 37.7%. Notably, the diversity of these mutations was observed between the SB-728-T product and samples at 3-4 years of age, and CD45RA int RO int T SCM These cells remained unchanged, suggesting that they most likely represent the long-lived memory subset contributing to the long-term polyclonal persistence of CCR5 gene-edited cells. Furthermore, CD45RA at 3-4 yearsint RO int CD95- or CD45RA + T SCM Not the persistence of cells, but CD45RA int RO int T SCM The persistence of the mutation was significantly correlated with an increase in the total CD4+ T cell count (Table 1). In summary, these results suggest that SB-728-T injection is associated with the long-term persistence of the CCR5 gene mutation and the rearrangement of novel T cells. SCM This indicates that it leads to a subset. [Table 1]

[0215] T 3-4 years after injection EM The presence of CCR5 gene mutations in short-lived memory cells such as (Figure 3B) indicates that T SCM and T CM CCR5 gene-edited cells within long-lived memory cells such as T EM It differentiates into CCR5 gene-edited T cells, and the CCR5 gene-edited T cells continue to be used until 3-4 years after injection. EM This suggests that it leads to the maintenance of a small subset of cells. CD45RA for HIV reservoir decay int RO int T SCM To investigate the role of CD45RA, we first quantified the levels of integrated HIV DNA in SB-728-T products and 3-4 year old samples in CD4+ T cell subsets. int RO int T SCM The cells were found to have significantly lower levels of integrated HIV DNA compared to other memory subsets (1.99 log10, 95% CI: CD45RA at 3-4 years). int RO int T SCM Middle 1.64-2.34 vs 2.8 log10, 95% CI;T CM Medium 2.48~3.13, P=0.016, 2.79 log10 95% CI:T TM Medium 2.31~3.28, P=0.016, 2.87 log10 95% CI:TEM (Intermediate 2.28~3.45, P=0.023). Furthermore, CD45RA int RO int T SCM Only 5.3% of cells contributed to CD4+ T cells in samples from year 3–4 (Figure 3c), but other memory subsets contributed to a significantly higher frequency of cells in the pool of cells with HIV tot DNA [T CM In this case, 45.5% (P=0.0078), T TM In the case of 16.5% (P=0.0156), and T EM In this case, the result was 29.6% (P=0.0156).

[0216] CD45RA possessing low levels of integrated HIV DNA int RO int T SCM The differentiation of cells into other memory subsets may provide a mechanism underlying the decay of the HIV reservoir in total CD4+ T cells. To investigate this, we constructed a sparse linear multivariate model to predict the change in the frequency of PBMCs carrying total HIV DNA after injection and CD45RA int RO int T SCM Cell count, CD45RA int RO int T SCM Frequency of pentamer replication in cells, and CD45RA int RO int T SCM and T EM The number of mutations shared between them was included as a possible independent variable. Our analysis showed that greater decay in the HIV reservoir after infusion corresponds to higher CD45RA at 3-4 years. int RO int T SCM Cell count (P=0.0018), CD45RA in 3-4 years. int RO int T SCM Higher frequency of pentamer replication in (P=0.005), and CD45RA int RO int T SCM The frequency of pentamer replication in and T in the 3rd to 4th year EMThis was best predicted by a lower ratio of the frequency of pentamer replication in ((P=0.0014)(adjusted R 2 =0.99, F-test: P=0.0008, Figure 3D). These results are from CD45RA int ROintT SCM Long-term persistence of cell numbers and T EM We demonstrated that maintaining a subset of CCR5 gene-edited cells within a population is important for reducing HIV reservoirs, and CD45RA int RO int T SCM This suggests that cells can differentiate into a pool of more differentiated memory cells, and that this pool can be replenished with a detectable proportion of cells possessing the CCR5 gene-editing allele that makes them resistant to infection.

[0217] CD45RA int RO int T SCM These cells express genes associated with quiescence and self-regeneration, and can differentiate into other memory subsets. CD45RA int RO int T SCM Our results demonstrating their effects on the persistence and long-term CD4+ T cell rearrangement suggest that these cells express genes and pathways that confer long-term persistence. Transcriptional analysis of the selected CD4+ T cell subset was performed on samples 3–4 years after injection. Multidimensional scaling of gene expression changes was used for CD45RA int RO int T SCM CD45RA + T SCM Rather, T CM and T EM It showed a greater difference between the two. In addition, CD45RA + T SCM to T EM Compared to T, CM and CD45RA + T SCM Compared to the standard, a larger number of differentially expressed genes (DEGs) were found (5022 vs 2943, vs 2136). Gene set enrichment analysis (GSEA) was performed on CD45RA.int RO int T SCM The cells were enriched with genes involved in stem cell properties (such as the Notch signaling pathway required for the maintenance of undifferentiated HSCs via Wnt), as well as metabolic pathways contributing to cell persistence, such as fatty acid oxidation, oxidative phosphorylation, and pyruvate metabolism. CM and T EM Compared to both (Figure 4A), genes associated with apoptosis, effector function, cell cycle, and JAK-STAT signaling were downregulated, and CD45RA int RO int T SCM This suggests that the cell is in a more quiescent state than other memory subsets.

[0218] CD45RA differentiates into other memory subsets int RO int T SCM To evaluate its capabilities, CCR5 ZFN-mediated mutations were sequenced in a selected CD4+ T cell subset, and CD45RA in the SB-728-T product was analyzed. int RO int T SCM Cell-specific sequences were identified (n=3,881). Distribution analysis of these sequences 3–4 years post-injection in various CD4+ T cell memory subsets (Figure 4B) shows CD45RA int RO int T SCM Unique mutations lead to short-lived T EM The results showed that it was detected in all memory T cell subsets, including (0.49% (95% CI: 0%~1.36%)). These results indicate that CD45RA int RO int T SCM This demonstrates that cells can differentiate into a pool of more differentiated memory cells and replenish that pool. CD45RA compared to other memory subsets. int RO int T SCMTo further characterize the differentiation state, their polyfunctional responses after stimulation with anti-CD3 / 28 coated beads (Figure 4c), SEB, or PMA / ionomycin were examined by flow cytometry. Our analysis showed CD4+ CD45RA int RO int T SCM T CM T TM , and T EM It was considered less differentiated than CD45RA. int RO int T SCM The undifferentiated state of cells was confirmed by analyzing the expression levels of transcription factors associated with the involvement of Th1 (T-bet and Eomes), Th2 (GATA-3), and Th17 (RORgt) lineages. (Naive cells and CD45RA) + T SCM Similarly, CD45RA int RO int T SCM The cells did not express Th-specific transcription factors (Figure 3d). Further analysis of immune checkpoint markers is needed. int RO int CD95 + Cells, T TM and T EM It has shown significantly lower levels of PD-1, TIGIT, and SLAM than CD45RA. int RO int CD95 + This suggests that the cells are less likely to be purged than other memory subsets (Figure 28B). In summary, these results suggest that CD45RA int RO int T SCM However, it exhibits stem cell characteristics including lifespan and pluripotency, T CM , T EM and T EM This indicates that these are more differentiated progenitor cells than memory cells.

[0219] Next, CD45RA int RO int T SCM and CD45RA + TSCM We compared the cellular transcriptomes to investigate differences in gene expression and specific pathways involved in self-renewal, including the Wnt signaling cascade, a pathway implemented to maintain the "stem-like" phenotype of T cells (Figure 4F). Multidimensional scaling of gene expression variance was used to characterize CD45RA, which was previously described. + CD95 + T memory stem cells (T SCM ) However, the CD45RA explained in this study int RO int T SCM The cells demonstrated transcriptional differences from the population (Figure 4E). A closer examination of the cutting-edge genes combined from these gene sets revealed upregulation of the expression of several genes crucial for maintaining stem cell characteristics. These include Wnt factors and their receptors (Frizzled-FZD), known to initiate the stem cell maintenance cascade, as described above (Figure 4G). This was combined with a downstream signaling cascade including DVL, β-catenin, and TCF7 genes, known to prevent effector T cell differentiation. Further downstream mechanisms of stem cell characteristics, defined by the upregulation of SOX genes, were also observed (Figure 4g). Interestingly, CD45RA int RO int T SCM Those CD45RA + T SCM Compared to the counterpart, enrichment was observed in pro-inflammatory (MAPK, Jun, NFAT) and apoptotic (PSM) gene sets (Figure 4G). The enhancement of the pro-inflammatory signature in this subset may indicate an increased ability to differentiate into more "effector" cells. Interestingly, the cutting-edge gene combinations associated with the Wnt signaling cascade were positively correlated with a long-term increase in CD4+ T cell count and negatively correlated with a reduction in the HIV reservoir after infusion.

[0220] In summary, these results indicate that CD45RA int RO int T SCMThe phenotypic cells possess the characteristics of long-lived, undifferentiated memory cells, representing a novel and distinct T gene. SCM Verify that it constitutes a subset.

[0221] CCR5 gene edited CD45RA int RO int T SCM The frequency of this correlates with viral load control in participants who experienced a 6-week treatment interruption after SB-728-T infusion. Next, CD45RA int RO int T SCM The impact of infusion of CCR5 gene-edited CD4+ T cells containing SB-728-T on the control of viremia at ART discontinuation was evaluated. Samples were analyzed from an independent clinical trial (SB-728-1101 study, n=15, 5 cohorts; see materials and methods) in which participants underwent analytical treatment discontinuation (ATI) 6 weeks after infusion of the SB-728-T product. Analysis of viral load levels showed that viral loads during ATI (week 22) were significantly lower than previous pre-ART viral load setpoints (P=0.0067) (Figure 5A), suggesting that infusion of the SB-728-T product may have led to transient but incomplete control of viremia in the majority of participants. Six individuals who extended ATI showed viral load measurements of less than 10,000 copies / mL and CD4+ T cell counts greater than 500 cells / μl at week 22, and subsequently spent 0.5–2 years in ATI. At that time, the 12-month viral load of the five participants who were undergoing ATI ranged from 130 to 16,000 copies / mL. One of these individuals (01-060) had the protective human leukocyte antigen (HLA) allele, HLA-B57.

[0222] Coincidentally, the significant reduction in viral load at week 22 compared to previous pre-ART viral load setpoints was significantly correlated with a large change in CD4+ T cell count at peak cell proliferation (Figure 5B) and a higher frequency of CCR5 gene-edited alleles pre-ATI (week 6) (Figure 5C). These results highlight the link between CCR5 gene-edited cell proliferation and viral load control during SB-728-T injection, and were also shown to be enriched in CCR5 gene mutations in the 1101 study (Figure 5d, e), indicating a link between T in post-ATI viral load reduction. SCM This suggests the role of subsets. Correlation analysis between viral load and CD4+ T cell subset number showed higher CD45RA before ATI (week 6). int RO int T SCM and CD45RA + RO - T SCM Cell number (especially CCR5 gene editing T SCM Only cell count correlated with a significant reduction in viral load at week 22 compared to previous viral load setpoints (Figure 5F, G).

[0223] Next, we investigated the functional response of HIV-specific CD8+ T cells, which have been previously shown to play a crucial role in regulating viral replication (REFS) after ATI, and examined the peak frequencies of cytokine (IFN-γ, TNF-α, IL-2) production by HIV-specific CD8+ T cell subsets after ATI, as well as CD45RA pre-ATI (week 6). int RO int T SCM We constructed a multivariate regression model to predict the change in viral load at week 22 compared to historical pre-ART viral load setpoints using a number of cells. Our analysis showed that a reduction in viral load at week 22 relative to historical pre-ART setpoints corresponds to IL-2-producing CD8+ T TM Along with the peak frequency of cells, higher CD45RA before ATI int RO int T SCM Cell number best predicts and explains 95% of changes in viral load (Figure 5H). These results suggest that decay in the HIV reservoir is related to CD45RA int ROint T SCM Cells (p=0.05) and CD8+ T cells producing IL-2 TM We demonstrate a significant and negative association with cell proliferation (P=0.02).

[0224] CCR5 gene editing T EM The frequency of this correlates with viral load control in participants who experienced treatment interruption during the 6-week SB-728-T infusion. CCR5 gene edited CD45RA int RO int T SCM To test the hypothesis that cells contribute to the regulation of viral load through differentiation into other memory subsets in ATI, CD45RA int RO int T SCM We first identified product-specific CCR5 mutations and tracked their persistence in other memory cells post-injection and post-ATI. Our results were CD45RA int RO int T SCM Product-specific CCR5 mutations were detected in all memory subset CD4+ at weeks 6 and 22 (frequency numbers added, Figure 6A), and CCR5 gene edit CD45RA int RO int T SCM It emphasizes the ability of cells to differentiate.

[0225] To investigate the long-term effects of viral replication on the persistence and differentiation ability of CCR5 gene-edited CD4+ T cell memory subsets, we then modeled the homeostasis of CCR5 gene-edited and unedited CD4+ T cell subsets during viremia (weeks 6–month) in five individuals with extended ATI beyond 12 months using a standard differential equation model. Using individual fitting routines in Monolix, we obtained mortality (γ), proliferation (ρ), and transition (φ) rates (cells / day) for each CCR5 gene-edited and unedited CD4+ T cell subset. We then developed CCR5 gene-edited CD45RA + T SCM CD45RA intRO int T SCM , and T CM We observed that the mortality rate of memory CD4+ T cells was, on average, three times lower than that of the unedited counterpart (Figure 6B). In addition, CCR5 gene-edited CD45RA + T SCM CD45RA int RO int T SCM , and T CM The mortality rate of memory CD4+ T cells was lower than their transition rate (Figure 6B). Furthermore, using a sensitivity analysis test in Matlab to identify the relationship between parameters and cell number, the model parameters for transition were found to be lower for CCR5 gene-edited CD45RA after ATI. + T SCM CD45RA int RO int T SCM , and T CM We observed a significant negative correlation with cell number (Figure 6B). In summary, these results suggest that the presence of CCR5 mutations provides a protective effect on the CD4+ early memory subset, and that the time-dependent loss of these cells is far more likely to be due to differentiation of these cells than to cell death. In addition, sensitivity analysis also showed that CCR5 gene edit CD45RA int RO int T SCM The proliferation rate of T EM It also showed a significant positive correlation with the cell count of all CCR5 gene-edited cells, including CD45RA, which was not observed for the proliferation rate of other subsets (Figure 6B). int RO int T SCM This suggests that self-renewal is important for supplementing and maintaining CCR5 mutations in other memory subsets.

[0226] Supporting these findings, higher CCR5 gene-edited CD45RA levels were observed before ART discontinuation (week 6). + RO - T SCM CD45RA int RO int TSCM and T CM The number of cells increased after ATI (week 22) with more CCR5 gene-edited T cells. EM The cell count correlates with viremia. SCM T EM We confirmed that it can induce gradual differentiation into cells (Figure 6C-E).

[0227] T EM Cells have been shown to express the highest levels of CCR5 compared to other memory cells, suggesting that T is involved in viral replication. EM Maintaining a subset of CCR5 gene-edited cells within a subset may lead to protection from de novo infection. To investigate this, we measured the size of the HIV reservoir (estimated by integrated HIV DNA levels) in selected CD4+ T cell subsets at baseline and at 6 and 22 weeks post-infusion. Our results suggest that T cells carrying integrated HIV DNA... EM The frequency of T did not change significantly during ATI (Figure 6F). Close investigation revealed that 50% of the analyzed participants experienced T between weeks 6 and 22. EM It was revealed that the size of the reservoir in the cells increased, and the other half did not show any change or decrease in the frequency of integrated HIV DNA. Importantly, T in ATI EM Changes in the frequency of cells possessing integrated HIV DNA within T EM It was inversely correlated with the frequency of the CCR5 gene-editing allele in the population (Figure 6G). EM It was further found that a higher frequency of the CCR5 gene-edited allele at week 22 in a subset (not other subsets) was specifically correlated with a significant reduction in viral load at week 22 compared to previous pre-ART viral load setpoints (Figure 6H-I). In summary, these results suggest that CCR5 gene-edited T EM Continuous cell replenishment, as a result of differentiation from those progenitor cells, leads to T in viremia. EM Limiting the size of the reservoir inside, and T EM This study demonstrates that superior protection from de novo infection of a subset of viruses influences the control of active viral replication during ATI.

[0228] Observations from this study indicate a novel CD45RA int RO int T SCM Further supporting our findings generated from the SB-728-0902 cohort, we demonstrate that a subset possesses the highest levels of CCR5 gene-editing alleles and can differentiate into other memory subsets. Short-lived T1s expressing CCR5 mutations EM Differentiated memory T cells, including these, are protected from viral infection, which leads to the control of viremia and the progressive decay of the HIV reservoir, as observed in both studies.

[0229] CD45RA+ T SCM The subsets have been previously described along with the characteristics of conventional memory T cells, enhanced self-renewal, and the ability to differentiate into other memory subsets. CD45RA+CD45RO+CCR7+CD27+CD95+T SCM Phenotypes like the one described above have been previously reported after in vitro proliferation of purified CD4+ and CD8+ naive T cells co-stimulated in the presence of cytokines such as IL-2, IL-7, IL-15, or IL-21, but the ability of these cells to survive in vivo and whether some of these cells can revert to the CD45RA+CD45RO- phenotype had not been investigated. In addition, CD4+ subsets expressing low levels of CD45RA and CD45RO appear to emerge in vivo at the initiation of ART in combination with IL-2 therapy, which correlates with CD4+ T cell increase, and CD45RA int RO int We also demonstrate that cells can be generated in vivo in response to homeostasis. The CD45RA we identified in this study... int RO int T SCM The subset also demonstrated the ability to self-renew, as observed by gene set enrichment of the Wnt signaling cascade; upregulation of additional stem cell-like (SOX gene) mechanisms; and CD45RA+ T SCM Unlike the subset, CD45RA int ROint T SCM Further observation of the enhancement of pro-inflammatory and apoptotic signatures specific to the subset, and these T SCM The distinctions between groups were further emphasized.

[0230] T SCM It has been previously shown that this can tolerate HIV infection. The importance of limiting HIV infection in early memory cells to maintain CD4+ T cell homeostasis has been shown not only in non-human primates but also in individuals with non-progressive viral HIV infection. (Enriched in the CCR5 gene-edited allele in secondary lymphoid tissue (up to 40% peripherally at 3-4 years)) SCM The presence of cells (even if HIV replication is not completely inhibited, but partially inhibited) may lead to their long-term survival. 。 This, in turn, improves adaptive immune function overall, increases CD4+ T cell count, controls HIV and other pathogens, and consequently reduces reservoir size, as shown in current studies. In fact, we found that in the third to fourth year, T TM and T EM In short-lived cells such as T in SB-728-T products SCM We detected CCR5 gene-edited cells specific to the subset, and T SCM T EM We confirmed the differentiation potential of CCR5 gene-edited CD4+ T cells. Furthermore, these observations support the modeling of homeostasis in the CCR5 gene-edited CD4+ T cell memory subset, and a reduction in mortality was observed. In addition, biphasic decay analysis of HIV DNA ruled out the possibility that dilution was the cause of HIV decay. However, our multivariate model showed that CCR5 gene-edited CD45RA int RO int T SCM We demonstrated that the long-term persistence of this contributes to the decay of the HIV reservoir after infusion.

[0231] The central hypothesis was that providing protection from HIV infection to a small subset of T cells would offer a comprehensive benefit and enable control of viral replication. In support of this, the results of both SB-728-0902 and SB-728-1101 studies confirmed the role of SB-728-T injection in restoring T cell homeostasis and in generative assistance to HIV-specific CD8 T cells. SCM T, which protects against HIV infection EM This can lead to differentiation into CD45RA. The kinship assistance provided is further emphasized by the observed decay in HIV reservoirs. int RO int T SCM Cells and GAG-specific CD8+ T TM Both IL-2-producing cells were negatively correlated with proliferation. The role of IL-2 production by HIV-specific CD8+ T cells in viral replication has been previously demonstrated.

[0232] Our results show that CCR5 gene edited and unmodified T in injected products SCM We recognized that both cell types could be due to ex vivo proliferation, and that such cells are protected in vivo by ART. Supporting this hypothesis, we observed significant proliferation of both CCR5-modified and unmodified cells after injection. Interestingly, our model is CD45RA int RO int T SCM This shows that the proliferation rate of all CCR5 gene-edited cells was positively correlated with the cell count. However, it should be noted that previous studies using adoptively transferred anti-CD3 / CD28 cost-stimulated CCR5-unmodified cells failed to sustainably increase the CD4+ T cell count in HIV+ participants. Currently, a randomized clinical trial is underway in which CCR5-modified and unmodified cells will be injected to address this issue more clearly.

[0233] The results described here demonstrate that a single infusion of CCR5 gene-edited cells is safe, well-tolerated, and may lead to a significant reduction in HIV DNA levels (and potentially a reproducible HIV reservoir). In addition, the long-term persistence of gene-edited memory stem cells allows dysfunctional / infected old memory cells to be replaced by new, infection-protected cells, thereby promoting immune system regrowth. The non-invasive and autologous aspects of this therapy make it more accessible and less burdensome than hematopoietic stem cell transplantation. Advances in zinc finger nuclease mRNA delivery via electroporation are expected to enable multi-dose regimens and significantly improve CD4+ T cell counts. In line with this, multiple infusions of unmodified anti-CD3 / CD28 cost-stimulated unmodified CD4+ T cells every 8 weeks have previously resulted in a significant increase in cell counts one year after infusion. In addition, as the CD4+ T cell count increased after each infusion, products from HIV+ subjects with a high CD4+ T cell count were shown to proliferate better in vitro compared to subjects with a low CD4+ T cell count; therefore, products produced thereafter are also expected to result in better engraftment and persistence. Furthermore, statistical analysis of HIV DNA results showed that the reservoir was partially reduced by 6 weeks of infusion, suggesting that optimal viral load control can be achieved if treatment is discontinued after an extended period along the second phase of decay.

[0234] In summary, our results demonstrate that injecting CCR5 gene-edited cells provides a unique therapeutic intervention that improves T cell homeostasis and reduces total HIV reservoir. Theoretically, combining this approach with other interventions may further improve outcomes.

[0235] While the present invention has been shown and described with particular reference to its preferred embodiments, those skilled in the art will understand that various modifications of form and detail can be made without departing from the scope of the invention as contained in the appended claims. All patents, publications and references cited in the foregoing specification are incorporated herein by reference in their entirety.

Claims

1. A method for generating an enriched population of CD4 / CD8 T cells lacking functional CCR5 and / or CXCR4 HIV co-receptors, wherein the method is: To isolate CD4 / CD8 T cells from the target biological sample, CD4 / CD8 T cells to CD45RA ｉｎｔ CD45RO ｉｎｔ To isolate a population of CD4 / CD8 T cells exhibiting the phenotype, CD45RA ｉｎｔ CD45RO ｉｎｔ Before isolating the population of phenotypic CD4 / CD8 T cells, modify the CD4 / CD8 T cells so that they lack functional CCR5 and / or CXCR4 HIV co-receptors, and / or the CD45RA ｉｎｔ CD45RO ｉｎｔ CD4 / CD8 T cells exhibiting the phenotype lack functional CCR5 and / or CXCR4 HIV co-receptors, as described above. ｉｎｔ CD45RO ｉｎｔ A method comprising modifying a population of isolated CD4 / CD8 T cells having a specific phenotype.

2. The method according to claim 1, wherein the biological sample comprises peripheral blood mononuclear cells isolated from the subject.

3. The method according to claim 1, wherein the isolated T cells are CD4+ T cells.

4. The method according to claim 1, wherein the isolated T cells are CD8+ T cells.

5. The method according to claim 3, wherein the isolated CD4 / CD8 T cells express at least one of CD95, CD127, or CD27.

6. The method according to claim 3, wherein the isolated CD4 / CD8 T cells intermediately express 4-1BB.

7. The method according to claim 3, wherein the isolated CD4 / CD8 T cells express at least one of IL17RA, CD5, IL2RG, IGF2R, SLC38A1, IL7R, SLC44A2, SLC2A3, CD96, CD44, CD6, CCR4, IL4R, or SLC12A7.

8. The isolated CD4 / CD8 T cells are CD45RA ｉｎｔ CD45RO ｉｎｔ The method according to claim 1, having a CD95+CD127+CD27+ phenotype

9. The isolated CD4 / CD8 T cells are CD45RA ｉｎｔ CD45RO ｉｎｔ The method according to claim 1, having CD95 + CD127 + CD27 + IL7R + CD44 + SCL38A1 + IL2RG + CD6 + CD5 + phenotype.

10. The method according to claim 1, further comprising activating the isolated CD4 / CD8 T cells with an anti-CD3 antibody and / or an anti-CD28 antibody.

11. The isolated CD4 / CD8 T cells are treated as CD45RA ｉｎｔ CD45RO ｉｎｔ The method according to claim 12, further comprising culturing with amounts of IL7 and IL15 effective in promoting the expansion and / or formation of an enriched population of phenotypic CD4 / CD8 T cells.

12. CD45RA ｉｎｔ CD45RO ｉｎｔ To maintain the phenotype, the CD45RA in the culture medium containing TGFβ / IL-1β ｉｎｔ CD45RO ｉｎｔ The method according to claim 12, further comprising culturing the isolated CD4 / CD8 T cells having the phenotype.

13. The method according to claim 1, wherein the CD4 / CD8 T cells lacking the functional CCR5 and / or CXCR4 HIV co-receptors are modified by inactivating the genes encoding CCR5 and / or CXCR4.

14. The method according to claim 13, wherein the isolated T cells are genetically modified by at least one of transduction, transfection, and / or electroporation.

15. An isolated, enriched CD4+ T cell population, wherein the T cell population comprises CD45A and CD45O (RA ｉｎｔ RO ｉｎｔ An isolated, enriched CD4+ T cell population characterized by the co-expression of intermediate cells on the surface and the expression of CD95+, CD127+, and CD27+, wherein the cells are modified to lack functional CCR5 and / or CXCR4 HIV co-receptors.

16. The population according to claim 15, which, when administered to subjects infected with HIV, can promote at least one of the following in the subjects: a sustained increase in the absolute CD4 cell count, restoration of HIV-specific T cell immunity, and substantial decay in the HIV reservoir.

17. Intermediate cell surface co-expression of CD45A and CD45O (RA ｉｎｔ RO ｉｎｔ A population of enriched memory stem cells (Tscm) CCR5 and / or CXCR4 gene-edited CD4+ T cells characterized by the expression of CCR7+, CD27+, CD28+, CD95+, CD127+, CD58+, and CD95+, wherein the gene-edited cells lack the functional CCR5 HIV co-receptor.

18. A method for treating an HIV-infected subject, comprising administering to the subject a concentrated T cell population according to any one of claims 15 to 17.

19. The method according to claim 18, wherein the subject is receiving and / or continuing to receive antiretroviral therapy.

20. The method according to claim 10, wherein the enriched T cell population is administered to the subject in an amount effective in promoting at least one of the following: a sustained increase in the absolute CD4 cell count in the subject, restoration of HIV-specific T cell immunity, and substantial attenuation of the HIV reservoir.

21. A method for treating an HIV-infected subject, comprising CD45A and CD45O (RA ｉｎｔ RO ｉｎｔ A method comprising administering to the subject an enriched population of CCR5 and / or CXCR4 gene-edited CD4+ T cells characterized by the co-expression of intermediate cell surface genes.

22. The method according to claim 21, wherein the subject is receiving and / or continuing to receive antiretroviral therapy.

23. The method according to claim 22, wherein the enriched T cell population is administered to the subject in an amount effective in promoting at least one of the following: a sustained increase in the absolute CD4 cell count in the subject, restoration of HIV-specific T cell immunity, and substantial attenuation of the HIV reservoir.

Images