Long-lived T cells for treating HIV infection

Abstract

The method for treating an HIV-infected subject includes administering to a subject a CD45A and CD45O (RA) antibody. int RO int The method includes administering to a subject an enriched CCR5 and / or CXCR4 gene-edited CD4+ T cell population characterized by intermediate cell surface co-expression of CCR5 and / or CXCR4.

Description

[Technical Field]

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

[0002] The existence of a small pool of latently infected cells is a major obstacle to cessation of antiretroviral therapy (ART) and eradication of human immunodeficiency virus (HIV). While ART can durably suppress viral replication, HIV persists indefinitely, requiring infected individuals to continue complex antiretroviral drug regimens for life. ART's ability to reconstitute immune function is highly variable. A subset of individuals (up to 45%) fails to demonstrate complete recovery of CD4+ T cell counts, even after years of effective ART. Impaired CD4+ T cell recovery has been associated with many host- and HIV-related factors, including impaired thymus formation and homeostasis. Because low CD4+ T cell counts in individuals on ART are associated with a higher risk of cancer and other diseases, novel therapeutic approaches to enhance immune function in such individuals are needed.

[0003] Studies modeling the latent HIV reservoir have shown minimal decay of total and integrated HIV DNA 4 years after ART initiation, particularly in individuals in whom ART was initiated during the chronic phase of infection. Several mechanisms contribute to HIV persistence, including homeostatic proliferation, dysfunctional host clearance mechanisms, and "latent" infection in long-lived memory CD4+ T cells, likely maintained by residual viral replication. Interestingly, all of these mechanisms are exacerbated in immunological non-responders and are associated with larger HIV reservoir sizes. Therefore, enhancing CD4+ T cell recovery may contribute to reducing the HIV reservoir during ART.

[0004] CCR5 is one of the major coreceptors for HIV entry. The therapeutic concept of providing a CCR5-deficient immune compartment to HIV-infected subjects was based on the transfer of CD34 from homozygous CCR5Δ32 matched donors. + This has been demonstrated in the "Berlin patient," who remained HIV-free since receiving an allogeneic bone marrow transplant of stem cells. While these results are promising, less invasive and more broadly applicable treatment strategies would be desirable. One approach is to reconstitute immune function through the adoptive transfer of autologous T cells, which has been successfully deployed in other viral infections, including cytomegalovirus and Epstein-Barr virus, but has largely failed in HIV infection, in part because CD4+ T cells remain susceptible 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 the infusion was safe and well-tolerated and led to an increase in CD4+ T cell counts and a reduction in the HIV reservoir. Summary of the Invention

[0005] The embodiments described herein include CD45RA int CD45RO int CD4 T cells and CD8 T cells (CD4 / CD8 T cells) with the phenotype CD45RA int CD45RO intThe present invention relates to a long-lived enriched population of genetically modified and / or engineered CD4 / CD8 T cells with a phenotype and their use in treating latent HIV infection in HIV-infected subjects, particularly those receiving and / or continuing to receive antiretroviral therapy. A subset of CD4 / CD8 T cells has been found to possess the phenotypic and molecular attributes of long-lived pluripotent stem cells. Similar to other known stem cell populations, this subset population has a reduced metabolic profile (upregulation of fatty acid metabolism and oxidative phosphorylation, and downregulation of cell cycling pathways), retains the ability to self-renew, and can differentiate into effector cells. This subset is primarily characterized by intermediate co-expression of CD45RA and CD45RO (CD45RA int CD45RO int ) is characterized by CD45RA int CD45RO int The phenotype of CD4 / CD8 T cells can also express CD95 (Fas), CD127 (IL7R), and CD27. Addition of low doses of cytokines IL-7 and IL-15 results in CD45RA int CD45RO int High doses of cytokines IL-7 and IL-15 may lead to effector differentiation of the cells, whereas high doses of cytokines IL-7 and IL-15 may lead to the formation of enriched populations of CD4 / CD8 cells with the phenotype.

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

[0007] In some embodiments, CD4 / CD8 T cells can be genetically modified to lack functional CCR5 and / or CXCR4 HIV coreceptors, CD45RA int CD45RO int A method for generating an enriched population of CD4 / CD8 T cells having a phenotype includes isolating T cells from a subject's biological sample. The biological sample can include a T cell-containing sample, such as peripheral blood mononuclear cells, from the subject with HIV to be treated, i.e., autologous T cells from the subject to be treated. The isolated T cells can include CD4+ T cells and / or CD8+ T cells.

[0008] CD45RA int CD45RO int A population of CD4 / CD8 T cells having the phenotype can be separated from the isolated T cells. In some embodiments, the CD4 / CD8 T cells are CD45RA int CD45RO int Prior to separating the population of CD4 / CD8 T cells having the phenotype from the isolated T cells, the CD4 / CD8 T cells can be genetically modified to lack functional CCR5 and / or CXCR4 HIV coreceptors. In other embodiments, the CD45RA int CD45RO int A population of CD4 / CD8 T cells with the phenotype CD45RA was isolated after separation from isolated T cells. int CD45RO int A population of CD4 / CD8 T cells having the phenotype can be genetically modified to lack functional CCR5 and / or CXCR4 HIV coreceptors.

[0009] In some embodiments, the 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 in the cells.

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

[0011] In other embodiments, the isolated CD4 / CD8 T cells can 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.

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

[0013] In other embodiments, the method may include activating the isolated CD4 / CD8 T cells with anti-CD3 and / or anti-CD28 antibodies prior to genetic modification and / or isolation. The activated CD4 / CD8 T cells may be induced by CD45RA int CD45RO int Once isolated, the CD4 / CD8 T cells can be cultured with an amount of IL7 and IL15 effective to promote the expansion and / or formation of an enriched population of CD4 / CD8 T cells having the phenotype 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 methods described herein, wherein at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or 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 composition or enriched T cell population may have a phenotype. The composition or enriched T cell population can be administered to a subject with HIV infection to treat HIV infection. In some embodiments, administration of the composition or enriched T cell population to a subject with HIV can promote at least one of a sustained increase in absolute CD4 cell count, restoration of HIV-specific T cell immunity, and substantial attenuation of the HIV reservoir in the subject. In some embodiments, the subject has been receiving and / or continues to receive antiretroviral therapy. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a flow diagram showing a method for generating an enriched population of CD4 / CD8 T cells with a CD45RAintCD45ROint phenotype. [Figure 2A-F]Plots showing HIV reservoir decay after SB-728-T infusion correlate with persistence of CCR5 gene-edited cells. A, Boxplots with overlaid jitter showing the frequency of cells harboring total HIV DNA per 106 PBMCs at baseline (BL), 1 year, and 2 years post-infusion. Boxes show the median, first, and third quartiles, with whiskers extending to the maximum and minimum values. Individual data points are shown 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, The frequency of integrated HIV DNA copies per 106 purified CD4+ T cells is shown at BL and at years 2–3 (long-term follow-up). Participants in Cohorts 1, 2, and 3 are shown in blue, green, and red, respectively. *P<0.05, Wilcoxon rank-sum test. C-D, Association between change in frequency of PBMCs harboring total HIV DNA at long-term time points (ratio of log10 values ​​at day 720 to day 0) and pentamer replication and fold expansion of marked cells at 21 days (C) and 3-4 years (D) post-infusion. Scatterplots and predictions from the robust regression model are shown with 95% confidence intervals (shaded regions). E, Representative example of biphasic decay analysis of HIV DNA (participant 3-01) using Monolix, software for parameter estimation of nonlinear mixed-effects models. The blue line represents a biphasic exponential fit line for HIV DNA copies per 10 PBMCs (represented by a red star). These lines represent the fast and slow decays, respectively, and the plateau reached after the end of the slow-decay phase. The inset highlights two intersections representing the onset and end of the slow-decay phase. F, Representative example of estimated total HIV DNA per 106 PBMCs (red line) expected as a result of dilution after infusion (participant 3-01). Also shown are 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 cells marked with pentamer replication by 4). [Figure 3A-D]Graphs and plots showing 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 minimally contributes to the CD4+ T cell reservoir. A, Bar graphs showing the mean distribution of naive, TCM, TTM, TEM, and CD45RAintROint frequencies in CD4+ T cells at BL (7 days to 3 months before infusion, n = 9), early (14-28 days, n = 6), mid-term (4-7 months, n = 7), and long-term (3-4 years, n = 9) time points after infusion. *P < 0.05, **P < 0.01; Wilcoxon rank-sum test. B, Median frequencies of pentamer replication markers per 10 cells measured in sorted TCM, TTM, and TEM memory subsets at day 14–4 months (n=7 for all three subsets), months 6–8 (n=7, 7, and 6, respectively), months 11–12 (n=7, 7, and 6, respectively), and years 3–4 (n=7, 7, and 5, respectively), and in CD45RA+TSCM and CD45RAintROintTSCM at months 9–10 (n=6 and 5, respectively), months 11–12 (n=3 and 5, respectively), and years 3–4 (n=7 and 8, respectively) post-infusion. N / A = not done; limitations in cryopreserved PBMCs prevented quantification of TSCM subsets at earlier time points. C, Boxplots with overlaid jitter showing the contribution of each subset to the CD4+ T-cell HIV reservoir in samples from years 3 to 4 (n=8 due to limited cell availability). The boxes show the median, first, and third quartiles, and the whiskers extend to the maximum and minimum values. P values ​​from the Wilcoxon rank-sum test are indicated. D, Three-dimensional scatterplots showing the change in frequency of PBMCs carrying total HIV DNA after infusion as a function of CD45RAintROintTSCM cell counts from years 3 to 4 (ratio of log10 values ​​at day 720 to day 0), the frequency of pentamer replication in CD45RAintROintTSCM from years 3 to 4, and the ratio of the frequency of pentamer replication in CD45RAintROintTSCM from years 3 to 4 to the frequency of pentamer replication in TEMs. A sparse linear multivariate model was constructed to predict reservoir decay.The multivariate regression model predicting the highest reservoir decay included three features: CD45RAintROintTSCM cell count in years 3–4 (z-axis), log10 pentamer replication level in CD45RAintROintTSCM in years 3–4 (x-axis), and pentamer replication ratio in CD45RAintROintTSCM / TEM in years 3–4 (y-axis). Each dot in the scatter plot corresponds to a participant with a dot size proportional to the day 720 HIV DNA / BL ratio, with greater decay symbolized by a smaller dot size. [Figure 4A-G]Tables, graphs, and plots depicting CD45RAintROintTSCM cells demonstrate their distinct identity from previously identified CD45RA+TSCM cells. A, Heatmap of selected pathways significantly enriched in genes induced or repressed in CD45RAintROintTSCM compared to TEM and TCM in 3-4 year old samples (n=7). Color gradients indicate GSEA normalized enrichment scores (NES ranging from -4 to +5) of pathways enriched in genes induced or repressed 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 CD45RAintCD45ROintCCR7+CD27+ T cell subsets in the SB-728-T product at 3-4 years old (n=5). 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 frequency of IFN-g, IL-2, and TNF-a cytokines produced by CD4+ T-cell subsets 3–4 years after infusion in response to anti-CD3 / CD28 stimulation. Responses were averaged for each cell subset (n=6). Pie charts show the percentage of cells producing one, two, or three functions. Arcs identify cell populations positive for IL-2, IFN-gamma, and TNF-alpha. Bar graphs show the relative frequency of different combinations of cytokine production. D, Histograms showing the expression of transcription factors T-bet, Eomes, RORgt, and GATA-3 in CD4+ T-cell subsets 3–4 years after infusion (n=7). *P<0.05, Wilcoxon rank-sum test. E, Multidimensional scaling (MDS) plot using Euclidean distance to highlight transcriptome variance between the CD45RAintROintTSCM and CD45RA+TSCM subsets. One dimension explains 27% of the transcriptome variance between the two TSCM subsets.CD45RAintROintTSCM are shown in red, and CD45RA+TSCM are shown in green (n=7). F, Heatmap of pathways identified by gene set enrichment analysis (GSEA) that focus on WNT signaling (the Reactome) and are significantly enriched in CD45RAintROintTSCM compared to CD45RA+TSCM. Aggregate gene sets using the leading edge of predefined pathways revealed significant enrichment of these genes in the CD45RAintROintTSCM subset. The scale represents the NES score, and red and blue boxes indicate positive and negative enrichment, respectively. Columns represent the CD45RAintROintTSCM and CD45RA+TSCM subsets. G, Co-expression network highlighting leading-edge genes significantly enriched within the CD45RAintROintTSCM subset. Network connectivity and co-expression were inferred using the GeneMania algorithm. [Figure 5A-H]Plots showing the correlation of CCR5 gene-edited TSCM before ATI with viral load control are shown. A, Plot showing viral load (VL) values ​​at week 22 (corresponding to week 16 of ATI) and historical pre-ART viral set point values ​​obtained from participants' charts (data available for 14 of 15 participants from Study 1101 Cohorts 1-5). Participants with extended ATI are shown in red. P values ​​for Wilcoxon rank-sum tests are shown. B-C, Spearman rank correlation between change in VL at week 22 to historical pre-ART viral set point and change in CD4+ T-cell count during peak expansion (weeks 1-3 post-infusion) (B) and frequency of the "pentamer replication" marker per 106 PBMCs (C) before ATI (week 6). 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 in cohorts 3–5 for whom cryopreserved cells were available for analysis. d–e, Boxplots with overlaid jitter depicting the frequency of CCR5 gene-edited alleles, as 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). Boxes indicate the median, first, and third quartiles, with whiskers extending to the maximum and minimum values. Participants with prolonged ATI are shown in red. n = 7; +P < 0.05; Wilcoxon rank-sum test. F–G, Spearman rank correlations between change in VL from week 22 to past pre-ART viral setpoint and (f) CD45RAintROintTSCM and (G) CD45RA+TSCM before ATI (week 6). Participants with prolonged ATI are shown in red. Dashed lines represent 95% confidence intervals. (n=8; data for past pre-ART VL setpoints were missing for participants 01–060 who had prolonged ATI and were therefore not included in VL-related analyses.)H, Three-dimensional scatter plot showing change in VL as a function of CD45RAintROintTSCM cell count (w22 to past set point) and frequency of CD8+ TTM cells producing IL-2 after gag peptide pool stimulation (for each participant, using the time point with the greatest response, Tmax) (n=8). A multivariate linear regression model was constructed using CD45RAintROintTSCM count at week 6 and the frequency of CD8+ T cell subsets producing IFN-γ, TNF-α, and IL-2 cytokines after gag peptide pool stimulation post-infusion. The multivariate model best predicting change in VL included CD45RAintROintTSCM count at week 6 (P=0.05) and HIV-specific CD8+ TTM cells producing IL-2 at Tmax (P=0.02). [Figure 6A-I]Plots showing that levels of CCR5 gene-edited TEM during ATI correlate with viral load control and reduce TEM HIV reservoir reseeding. A, Boxplot (n=7) showing the percent of ZFN-induced CCR5 mutations uniquely present in CD45RAintCD45ROintCCR7+CD27+ (TSCM phenotype) in the SB-728-T product, as detected in CD4+ T cell subsets at weeks 6 and 22 post-infusion. Boxes indicate the median, first, and third quartiles, with whiskers extending to a distance of 1.5*IQR. Outliers are indicated by dots. B, Schematic showing the dynamics of CCR5 gene-edited CD4+ T cell dynamics (see Materials and Methods for full model details and assumptions). Model parameters are obtained by taking the geometric mean of five individual fitted results for five participants with extended ATI durations. Parameters listed in boxes indicate those that showed significant correlation (≥ ±0.5) with cell population size, as determined by sensitivity analysis performed in MATLAB using 100,000 bins. 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, Boxplot with overlaid jitter (n=7) showing the frequency of integrated HIV DNA in TEM cells at weeks 6 and 22 post-infusion. Boxes show the median, first, and third quartiles, with whiskers extending to the maximum and minimum values. Dots and lines are shown for all participants. Participants who extended ATI are shown in red. P values ​​from the Wilcoxon rank-sum test are shown. G-H, Spearman rank correlations between the frequency of CCR5 gene-edited alleles in TEMs during viremia (week 22) and the change in viral load from week 22 (16 weeks post-ATI) to the past pre-ART viral set point (G) and the change in frequency of TEM cells carrying intermediate HIV DNA from week 6 to week 22 (H). Participants with extended ATI are shown in red. Dashed lines represent 95% confidence intervals.n=6; data on past pre-ART VL setpoints were missing for participants 01–060 who were not included in the VL-associated analyses due to extended ATI. I, Spearman rank correlation between the change in frequency of TEM cells carrying intermediate HIV DNA from weeks 6 to 22 and viral load levels at week 22 (16 weeks post-ATI). Participants with extended ATI are shown in red. Dashed lines represent 95% confidence intervals. n=8. [Figure 7] Figure 1 shows a plot depicting the size of the HIV reservoir in SB-728-0902 study participants at baseline. Correlation between CD4+ T cell count at baseline (BL) and the level of integrated HIV DNA at BL measured in purified CD4+ T cells. Spearman's rho (ρ) test was used. Dashed lines represent 95% confidence intervals. [Figure 8A-E]This figure shows that a single infusion of SB-728-T led to a sustained increase in total CD4+ T cell counts, an improved CD4:CD8 ratio, and long-term persistence of CCR5 gene-edited cells. A, CD4+ T cell counts are shown at baseline (BL, 7 days before infusion), day 14, months 3, 6, and 12, and at long-term follow-up, including the last follow-up at years 2 and 3-4. Means are indicated by black lines. Wilcoxon signed-rank test *P<0.05, **P<0.01. For panels A-C, participants in cohort 1 (approximately 1E10 infused cells) are indicated by blue symbols, cohort 2 (approximately 2E10 infused cells) are indicated by green symbols, and cohort 3 (approximately 3E10 infused cells) are indicated by red symbols. B, CD4:CD8 ratios at BL, day 14, months 3, 6, and 12, and years 2 and 3-4 are shown. The mean is indicated by the black line. *P<0.05, **P<0.01; Wilcoxon signed-rank test. C, Fold expansion of pentamer replication and marked CD4+ T cells after infusion was estimated for all nine study participants during follow-up, as described in Materials and Methods. Gray areas represent data points with a fold change of less than 1. D, Boxplot with overlaid jitter of pentamer replication (a marker for gene-edited cells) per 10⁶ mononuclear cells from rectal biopsies after infusion. The boxplot shows the 75th percentile (top), median (solid line within the box), and 25th percentile (bottom). The whiskers are plotted from the minimum to the maximum value. E, Plot of pentamer replication marker per 10⁶ PBMCs (black circles) and mononuclear cells from lymph node biopsies (LNMCs, squares) after infusion for three individuals in whom the pentamer replication marker was quantified in LNMCs. [Figure 9A-C]Plots showing the characteristics of the SB-728-T product are shown. A, Levels of integrated HIV DNA in purified CD4+ T cells from pre-manufacturing leukapheresis samples (BL) and post-manufacturing (SB-728-T product). P values ​​from 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, CD4+ T cell subset frequencies observed in the SB-728-T product. Lines represent the mean and standard deviation. C, CCR5 gene-edited allele frequencies within CD4+ T cell subsets in the SB-728-T product as measured by DNA sequencing of various CCR5 ZFN-induced mutations. Lines represent the mean and standard deviation. *P<0.05, **P<0.01; Wilcoxon signed-rank test. [Figure 10A-F]Plots show that the frequency of CD58+CD95+ cells in the CD45RAintCD45ROint and CD45RA+CD45RO- subsets increased after infusion, contributing to the persistence of CCR5 gene-edited cells. A-B, Histograms showing the mean frequency of CD58- and CD95-expressing cells in the CD45RAintCD45ROintCCR7+CD27+CD127+CD28+ (CD45RAintROintTSCM, A) and CD45RA+CD45RO-CCR7+CD27+CD127+CD28+ (CD45RA+TSCM, B) subsets at BL (n=6), mid-term (6-8 months, n=5), late (9-11 months, n=5), and long-term (3-4 years, n=6) time points after infusion. Error bars represent standard deviation. *P<0.05, **P<0.01; Mann-Whitney test. C-D, The frequencies of CD58+CD95+ cells within the CD45RA+RO- and CD45RAintROint subsets at 3-4 years correlated with the estimated fold expansion of CCR5 gene-edited CD4+ T cells in PBMCs at long-term time points (number of CCR5 gene-edited alleles at 3-4 years relative to the number (dose) of CCR5 gene-edited alleles infused). E, Longitudinal analysis 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) cell counts at BL (up to 3 months pre-infusion), and mid-term (6 months), late (8-11 months), and long-term (3-4 years) post-infusion time points for six subjects who underwent BL analysis. F, Longitudinal analysis of CD45RAintROintCCR7+CD27+CD127+CD28+CD58+CD95+ (referred to as CD45RAintROintTSCM) and CD45RAintROintCCR7+CD27+CD127+CD28+CD58-CD95- (referred to as CD45RAintROintCD95-) cell counts at BL (up to 3 months before infusion), and at mid-term (6 months), late (8–11 months), and long-term (up to 44 months) postinfusion time points for six subjects who underwent BL analysis. [Figure 11]A plot showing that CD45RAintROintTSCM cells have higher levels of CCR5 gene-edited alleles compared to other memory subsets is shown. Boxplots overlaid with jitter of CCR5 gene-edited allele frequencies within sorted CD4+ T cell subsets 2-4 years post-infusion, as measured by DNA sequencing of various CCR5 ZFN-induced mutations. Lines represent the mean and standard deviation. Boxplots show the 75th percentile (top), median (solid line within box), and 25th percentile (bottom). Whiskers are drawn from minimum to maximum. P values ​​from the Wilcoxon signed-rank test are shown. [Figure 12A-B] Plots showing that CCR5 gene-edited T cells contribute to polyclonal, unbiased T cell reconstitution. A, Bar graphs showing TCR diversity in pre-manufacturing samples (CD4+ T cells purified from BL leukapheresis samples, n=4), SB-728-T product (n=4), and purified CD4+ T cells at 7 days (peak of CCR5 gene-edited cell proliferation) (n=3) and 7–9 months (n=4) post-infusion. TCR clonal diversity was measured using the Shannon entropy index. NA = sample not available at this time point. B, CCR5 gene-edited allele diversity in CD4+ T cell subsets from the SB-728-T product compared to subsets from a longer-term time point (3–4 years) post-infusion using the Shannon entropy index (n=8). Lines represent the mean and standard deviation. P values ​​from the Wilcoxon signed-rank test are indicated. [Figure 13A-B]Graphs showing that CD45RAintROintTSCM cells are a minor contributor to the total HIV reservoir. Sorted CD45RA-CD45RO+ subsets (TCM, n=9; TTM, n=8; TEM, n=9; and CCR7+CD27, n=9) and CD45RAintCD45ROint subsets (total CD45RAintCD45ROint, n=9; and CD45RAintCD45ROintCCR7+CD27+, n=9) in the SB-728-T product (A), as well as sorted CD45RAintCD45ROint subsets at 3–4 years post-infusion. Histogram showing the mean levels of integrated HIV DNA (log copies / 10 cells) in the CD45RA-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] Plots showing that CD45RAintROintTSCM cells constitute a distinct population from the previously described CD45RA+TSCM subset are shown. A, Multidimensional scaling (MDS plot) was used to highlight transcriptome variance among CD4+ T cell subsets 3-4 years post-infusion. Euclidean distance was used to represent dimensionality reduction of top variant genes based on ANOVA (analysis of variance, F-test, n = 3358 transcripts, P ≤ 0.05). One dimension explains 50% of the variance among CD4+ T cell subsets. Different subsets are represented by different symbols. n = 7 samples per cell subset. B, Bar graph showing the number of differentially expressed genes (DEGs, P < 0.05) between CD45RAintROintTSCM and CD45RA+TSCM, TCM, or TEM. [Figure 15A-B]Plots showing viral load for subjects in the 1101 study who underwent analytical treatment interruption (ATI) after SB-728-T infusion are shown. A, Summary of the 1101 clinical trial. Subjects received escalating doses of Cytoxan (CTX) preconditioning 2 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 restarted ART by week 22 and 6 subjects with prolonged ATI (VL remained below 10,000 copies / mL and CD4+ T-cell counts above 500 cells / μl). Red lines represent ART resumption. [Figure 16A-B] Plots showing the distribution of CD4+ T-cell subsets and cell counts after infusion (week 6) and after treatment discontinuation (week 22) in Study 1101 are shown. Frequency (A) and cell counts (B) of CD4 T-cell subsets (naive, CD45RA+ TSCM, CD45RAintROintTSCM, TCM, TTM, and TEM) are shown 6 weeks after infusion (pre-ATI) and 22 weeks after infusion (end of ATI) (n=9). P values ​​from the Wilcoxon signed-rank test are shown. [Figure 17] Figure 1 shows the frequency of CCR5 gene-edited cells in CD4+ T-cell subsets after ATI in Study 1101. The frequency of CCR5 gene-edited alleles, as determined by DNA sequencing, is shown for CD4+ T-cell subsets (CD45RA+ TSCM, CD45RAintROintTSCM, TCM, and TEM) at 6 weeks (pre-ATI), 22 weeks, 7 / 8 months, and 12 months (during ATI) post-infusion for participants whose ATI was extended to at least 12 months. DETAILED DESCRIPTION OF THE INVENTION

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[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 to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0019] As used herein, "about" when referring to a measurable value such as an amount, temporal duration, etc., is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1 from the specified value, where such variations are appropriate for practicing the disclosed methods.

[0020] As used herein, "activation" refers to the state of T cells that are sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with inducible cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells undergoing cell division.

[0021] The term "antibody" as used herein refers to an immunoglobulin molecule that can specifically bind to a specific epitope on an antigen. An antibody can be an intact immunoglobulin derived from natural or recombinant sources, or an immunologically active portion of an intact immunoglobulin. An antibody is usually a tetramer of an immunoglobulin molecule. The antibodies of the present invention can 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 term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may involve either antibody production or activation of specific immunocompetent cells, or both. A skilled artisan will understand that virtually any macromolecule, including any protein or peptide, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response will therefore encode an "antigen," as that term is used herein. Furthermore, a skilled artisan will understand that an antigen need not 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 elicit a desired immune response. Furthermore, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that antigens can be 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 "autologous" is meant to refer to any material derived from the same individual that is later reintroduced into that individual.

[0024] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0025] "Xenogeneic" refers to a graft derived from an animal of a different species.

[0026] As used herein, "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0027] The term "expression" as used herein 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" refers to a molecule, such as an antibody, that recognizes and binds to another molecule or feature but does not substantially recognize or bind to other molecules or features in a 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, for example, compounds that bind to, partially or completely block stimulation of, reduce, prevent, delay activation of, inactivate, desensitize, or down-regulate the stability, expression, function, and activity of proteins, genes, and mRNAs, e.g., antagonists.

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

[0031] The terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of a corresponding naturally occurring amino acid.

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

[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 containing a guide sequence, a tracr sequence, and a tracr mate sequence. The term "guide sequence" refers to a sequence of approximately 10 to 30 (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs within a guide RNA that identifies a 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 by conventional Watson-Crick or other non-conventional methods. The percent complementarity 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 (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Fully complementary" means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region 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 refers to two nucleic acids that hybridize under stringent conditions.

[0035] A "binding protein" is a protein that can bind to another molecule. A binding protein can bind, for example, to a DNA molecule (DNA-binding protein), an RNA molecule (RNA-binding protein), and / or a protein molecule (protein-binding protein). In the case of a protein-binding protein, it can bind to itself (forming a homodimer, homotrimer, etc.) and / or to one or more molecules of one or more different proteins. A binding protein can have multiple types of binding activity. For example, a zinc finger protein has DNA-binding, RNA-binding, and protein-binding activity.

[0036] A "zinc finger DNA-binding protein" (or binding domain) is a protein or domain within a larger protein that binds to DNA in a sequence-specific manner via one or more zinc fingers, which are regions of amino acid sequence within the 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 responsible for binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length and exhibits at least some 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 predetermined nucleotide sequence, for example, through manipulation of the recognition helix region of a naturally occurring zinc finger or TALE protein (by modifying one or more amino acids). Thus, engineered DNA-binding proteins (zinc fingers or TALEs) are proteins that do not occur in nature. A non-limiting example of a method for engineering DNA-binding proteins is design and selection. Designed DNA-binding proteins are proteins that do not occur in nature, and their design / composition is primarily based on rational criteria. Rational criteria for design include the application of substitution rules and computerized algorithms to process information from existing ZFP and / or TALE designs and information in databases that store binding data. See, for example, U.S. Patent Nos. 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 primarily results from empirical processes such as phage display, interaction traps, or hybrid selection. See, e.g., U.S. Patent Nos. 8,586,526, 5,789,538, 5,925,523, 6,007,988, 6,013,453, and 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 purposes of this disclosure, "homologous recombination (HR)" refers to a specialized form of such exchange that occurs, for example, during repair of double-strand breaks in cells via homology-directed repair mechanisms. This process is known variously as "non-crossover gene conversion" or "short tract gene conversion" because it requires nucleotide sequence homology, uses a "donor" molecule to template the repair of a "target" molecule (i.e., the molecule that experienced the double-strand break), and leads to the transfer of genetic information from the donor to the target. Without wishing to be bound by theory, such transfer may involve mismatch correction of heteroduplex DNA formed between the broken target and the donor, and / or "synthesis-dependent strand annealing," in which the donor is used to resynthesize the genetic information that will become part of the target and / or associated processes. Such specialized HR often results in alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

[0041] In the disclosed methods, one or more targeted nucleases (e.g., CRISPR / Cas) described herein create a double-stranded break in a target sequence (e.g., cellular chromatin) at a predetermined site, and a "donor" polynucleotide having homology to the nucleotide sequence of the break region can be introduced into the cell. The presence of the double-stranded break has been shown to promote the integration of the donor sequence. The donor sequence can be physically integrated, or alternatively, the donor polynucleotide can be used as a template to repair the break via homologous recombination, introducing all or part of the nucleotide sequence in the donor into cellular chromatin. Thus, a first sequence in cellular chromatin can be modified and, in certain embodiments, converted to a sequence present in the donor polynucleotide. Therefore, the use of the terms "replace" or "substitution" can be understood to refer to the replacement of one nucleotide sequence with another nucleotide sequence (i.e., sequence replacement in an informational sense), and does not necessarily require the physical or chemical replacement of one polynucleotide with another.

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

[0043] In certain embodiments of methods for targeted recombination and / or replacement and / or modification of sequences in a region of interest in cellular chromatin, the 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") can contain sequence that is homologous, but not identical, to a 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 that is homologous to a sequence in the region of interest exhibits approximately 80-99% (or any integer therebetween) sequence identity to the genomic sequence to be replaced. In other embodiments, for example, when only a single nucleotide differs between the donor sequence and the genomic sequence of more than 100 contiguous base pairs, the homology between the donor and genomic sequence is greater than 99%. In certain cases, the non-homologous portion of the donor sequence can contain sequence that is not present in the region of interest, thereby introducing new sequence into the region of interest. In these examples, the non-homologous sequence is generally flanked by 50-1,000 base pairs (or any integer value therebetween) or any number of base pairs greater than 1,000 that are homologous or identical to a sequence in the region of interest. In other embodiments, 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 in a cell by targeted integration of a donor sequence that disrupts expression of the gene of interest. Cell lines with partially or completely inactivated genes are also provided.

[0046] Furthermore, the targeted integration method described herein can also be used to integrate one or more exogenous sequences.The exogenous nucleic acid sequence can 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 control elements (e.g., promoters).In addition, the exogenous nucleic acid sequence can produce one or more RNA molecules (e.g., small hairpin RNA (shRNA), inhibitory RNA (RNAis), microRNA (miRNA), etc.).

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

[0048] A "cleavage half-domain" is a polypeptide sequence that, in combination with a second polypeptide (either the same 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] An "engineered cleavage half-domain" is a cleavage half-domain that has been modified to form an obligate heterodimer with another cleavage half-domain (e.g., another engineered cleavage half-domain). See also U.S. Patent Publication Nos. 2005 / 0064474, 2007 / 0218528, 2008 / 0131962, and 2011 / 0201055, which are incorporated by reference in their entireties.

[0050] The term "sequence" refers to a nucleotide sequence of any length, which may be DNA or RNA, linear, circular, or branched, and either single-stranded or double-stranded. The term "donor sequence" refers to a nucleotide sequence to be inserted into a genome. The donor sequence may be any length, for example, 2 to 10,000 nucleotides in length (or any integer value therebetween or greater), preferably about 100 to 1,000 nucleotides in length (or any integer value therebetween), and more preferably about 200 to 500 nucleotides in length.

[0051] A "homologous, non-identical sequence" refers to a first sequence that shares a 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 to, but non-identical to, the sequence of the mutant gene. In certain embodiments, the degree of homology between the two sequences is sufficient to allow homologous recombination between them using normal cellular mechanisms. Two homologous, non-identical sequences can be of any length, and their degree of non-homology can be as small as a single nucleotide (e.g., for correction of a genomic point mutation 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). Two polynucleotides containing homologous, non-identical sequences do not need to be the same length. For example, an exogenous polynucleotide (i.e., donor polynucleotide) of 20 to 10,000 nucleotides or nucleotide pairs 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 (polynucleotide or amino acid) can be compared by determining their percent identity. The percent identity of two sequences, whether nucleic acid or amino acid, is calculated by dividing the number of exact matches between the two aligned sequences by the length of the shorter sequence and multiplying by 100. Approximate alignment of nucleic acid sequences is provided by the local homology algorithm of 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, MODayhoff 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 percent identity of sequences is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application. Suitable programs for calculating percent identity or similarity between sequences are generally known in the art; for example, another alignment program is BLAST, used with default parameters.For example, BLASTN and BLASTP can be used using the following default parameters: Genetic 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 online. For the sequences described herein, the desired degree of sequence identity ranges from approximately 80% to 100% and any integer value therebetween. Typically, the percent 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 the polynucleotides under conditions that allow the formation of stable duplexes between homologous regions, followed by digestion with a single-strand-specific nuclease and sizing of the digested fragments. Two nucleic acid or two polypeptide sequences are substantially homologous to each other if the sequences exhibit at least about 70%-75%, preferably 80%-82%, more preferably 85%-90%, even more preferably 92%, even more preferably 95%, and most preferably 98% sequence identity over a defined length of the molecule, as determined using the above-described methods. As used herein, "substantially homologous" also refers to sequences that exhibit complete identity to a specified 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. See, e.g., Sambrook et al., supra; Nucleic Acid Hybridization: A Practical Approach, editors BD Hames and SJ Higgins, (1985) Oxford; Washington, DC; IRL Press).

[0054] The selective hybridization of two nucleic acid fragments can be determined as follows. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of the hybridization event between such molecules. Partially identical nucleic acid sequences will at least partially inhibit the hybridization of completely identical sequences to target molecules. The inhibition of the 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 carried out using various degrees of selectivity, for example, using conditions ranging from low stringency to high stringency. When low stringency conditions are used, the lack of non-specific binding can be assessed using secondary probes that lack even a partial degree of sequence identity (e.g., probes having less than about 30% sequence identity with the target molecule), such that in the absence of non-specific binding events, the secondary probe will not hybridize to the target.

[0055] "Chromatin" is the nucleoprotein structure that constitutes the cellular genome. Cellular chromatin is composed of nucleic acids, primarily DNA, and proteins, including histones and non-histone chromosomal proteins. The majority of eukaryotic cellular chromatin exists in the form of nucleosomes, where the nucleosome core contains 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. Histone H1 molecules are generally associated with the linker DNA. For the purposes of this disclosure, the term "chromatin" is intended to encompass all types of cellular nucleoproteins 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. The genome of a cell is often characterized by a karyotype, which is the collection of all chromosomes that make up the genome of the cell. The genome of a cell can contain one or more chromosomes.

[0057] An "episome" is a replicating nucleic acid, nucleoprotein complex, or other structure that contains nucleic acid that is not part of the chromosomal karyotype of a cell. Examples of episomes include plasmids and certain viral genomes.

[0058] "Accessible region" refers to the site in cellular chromatin where the target site present in nucleic acid can be bound by an exogenous molecule that recognizes the target site.Without wishing to be bound by a particular theory, it is believed that accessible region is a region that is not packaged into a nucleosome structure.The distinct structure of accessible region can often be detected by the sensitivity to chemical and enzymatic probes, for example, nucleases.

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

[0060] An "exogenous" molecule is one that is not normally present within a cell but can be introduced into the cell by one or more genetic, biochemical, or other methods. Its "normal presence within a cell" is determined with respect to the cell's particular developmental stage and environmental conditions. Thus, for example, a molecule that is present only during embryonic muscle development is exogenous with respect to adult muscle cells. Similarly, a molecule induced by heat shock is exogenous with respect to cells that have not been subjected to heat shock. Exogenous molecules can include, for example, a functioning version of a dysfunctional endogenous molecule or a dysfunctional version of a normally functioning endogenous molecule.

[0061] Exogenous molecules can be small molecules, such as those produced by combinatorial chemical processes, or macromolecules, such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, and polysaccharides, any modified derivatives of the above molecules, or any complex containing one or more of the above molecules. Nucleic acids include DNA and RNA, can be single-stranded or double-stranded, linear, branched, or circular, and can be of any length. Nucleic acids include those that can form duplexes as well as triplexes. See, for example, U.S. Patent Nos. 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, gyrases, and helicases. Thus, the term includes a "transgene" or "gene of interest" that is an exogenous sequence that has been introduced into a cell.

[0062] An exogenous molecule can be the same type of molecule as an endogenous molecule, such as an exogenous protein or nucleic acid. For example, an exogenous nucleic acid can include an infectious viral genome, a plasmid or episome introduced into a cell, or a chromosome not normally present in the cell. 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. An exogenous molecule can also be the same type of molecule as an endogenous molecule, but can be derived from a species different from that from which the cell is derived. For example, a human nucleic acid sequence can be introduced into a cell line originally derived from a mouse or hamster. Methods for introducing exogenous molecules into plant cells are known to those of skill 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 co-precipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer.

[0063] In contrast, an "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, endogenous nucleic acids can include chromosomes, mitochondrial genomes, chloroplasts or other organelles, or naturally occurring episomal nucleic acids. Additional endogenous molecules can include proteins, such as transcription factors and enzymes.

[0064] As used herein, the term "product of an exogenous nucleic acid" includes both polynucleotide and polypeptide products, e.g., both transcription products (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 covalently. The subunit molecules can be of the same chemical type or 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 fusion proteins described above). Examples of the second type of fusion molecule include, but are not limited to, fusions between a triple-helix-forming nucleic acid and a polypeptide, and fusions between a minor groove binder and a nucleic acid. A "fusion polypeptide" is a polypeptide containing a polypeptide or a portion thereof (e.g., one or more domains) fused or conjugated to a heterologous polypeptide. Examples of fusion polypeptides include immunoadhesins that combine a portion of a Cas protein with an immunoglobulin sequence, and epitope-tagged polypeptides that may include a Cas protein, e.g., or a portion thereof fused to a "tag polypeptide." The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, but is short enough so as not to interfere with the nuclease activity of Cas. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 6 and 60 amino acid residues.

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

[0067] For purposes of this disclosure, a "gene" includes a DNA region encoding a gene product (see below) as well as all DNA regions that regulate the production of a gene product, regardless of whether such regulatory sequences are adjacent to the coding and / or transcribed sequence. Thus, a gene includes, but is not limited to, promoter sequences, terminators, translational 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 control regions. An "engineered gene" refers to a gene that has been modified in some way to be non-identical to the wild-type gene. Modifications can be in the form of targeted deletions, insertions, and truncations. An engineered gene can contain coding sequences from two heterologous genes or can contain synthetic gene sequences. An engineered gene can also contain changes in the coding sequence that are silent in the protein sequence (e.g., codon optimization). An engineered gene can also include genes in which regulatory sequences have been modified.

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

[0069] " Regulation " of gene expression refers to the change in the activity of gene. Regulation of expression can include, but is not limited to, gene activation and gene suppression. Genome editing (for example, cutting, modifying, inactivating, random mutation) can be used to regulate expression. Gene inactivation refers to the reduction of gene expression compared with cells that do not contain the CRISPR / Cas system described herein. Therefore, gene inactivation can 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, to which it is desired to bind an exogenous molecule. Binding may be for the purpose of targeted DNA cleavage and / or targeted recombination. A region of interest may reside, for example, in a chromosome, episome, organelle genome (e.g., mitochondria, chloroplasts), or infectious virus genome. A region 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 a non-transcribed region either upstream or downstream of a coding region. A region of interest may be as small as a single nucleotide pair or up to 2,000 nucleotide pairs in length, 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 (eg, T cells).

[0072] The terms "functionally linked" and "functionally linked" (or "operably linked") are used interchangeably in reference to the juxtaposition of two or more components (such as sequence elements) that allow for the normal function of both components and the likelihood that at least one of the components will mediate a function exerted by at least one of the other components. Illustratively, a transcriptional regulatory sequence, such as a promoter, is operably linked to a coding sequence if it controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptional regulatory sequence is generally operably linked to a coding sequence in cis, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operably linked to a coding sequence, even if it is not adjacent to it.

[0073] With respect to a fusion polypeptide, the term "operably linked" can refer to the fact that each component, when linked to the other component, performs the same function as if it were not so linked. For example, with respect to 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 operably linked if, in the fusion polypeptide, the Cas DNA-binding domain portion is capable of binding to its target site and / or its binding site, while the activation domain is capable of upregulating gene expression. With respect to 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 operably linked if, in the fusion polypeptide, the Cas DNA-binding domain portion is capable of binding to its target site and / or its binding site, while the cleavage domain is capable of cleaving 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 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 can have more, fewer, or the same number of residues as the corresponding native molecule and / or can contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well known in the art. Similarly, methods for determining the function of a protein 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 assays. 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 assays, or both genetic and biochemical complementation. See, e.g., Fields et al. (1989) Nature 340:245-246, U.S. Patent No. 5,585,245 and PCT WO 98 / 44350.

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

[0076] "Reporter gene" or "reporter sequence" refers to any sequence that produces a protein product that is easily measured, preferably, though not necessarily, by routine assay. 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 or fluorescent or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins that mediate enhanced cell growth and / or gene amplification (e.g., dihydrofolate reductase). Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA, or any detectable amino acid sequence. "Expression tags" include sequences encoding reporters that can be operably linked to a desired gene sequence to monitor expression of the gene of interest.

[0077] The terms "subject" and "patient" are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, dogs, cats, rats, mice, and other animals. Thus, the term "subject" or "patient" as used herein refers to any mammalian patient or subject to which 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] The term "therapeutic" as used herein means treatment and / or prophylaxis. A therapeutic effect is achieved by suppressing, ameliorating, or eradicating a disease condition.

[0079] The term "therapeutically effective amount" refers to an amount of a compound of interest that will elicit the biological or medical response in a tissue, system, or subject that is desired by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes a compound in an amount sufficient, when administered, to prevent or alleviate to some extent one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated. As used herein, "treating" a disease means reducing 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" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. A cell includes the primary subject cell and its progeny.

[0081] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into 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. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This 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, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0082] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0083] The embodiments described herein include CD45RA int CD45RO int CD4 T cells and CD8 T cells (CD4 / CD8 T cells) with the phenotype CD45RA int CD45RO int The present invention relates to a long-lived enriched population of genetically modified and / or engineered CD4 / CD8 T cells with a phenotype and their use in treating latent HIV infection in HIV-infected subjects, particularly those receiving and / or continuing to receive antiretroviral therapy. A subset of CD4 / CD8 T cells has been found to possess the phenotypic and molecular attributes of long-lived pluripotent 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 cell cycling pathways), retains self-renewal capacity, and can differentiate into effector cells. This subset is primarily characterized by intermediate co-expression of CD45RA and CD45RO (CD45RA int CD45RO int ) is characterized by CD45RA int CD45RO int The phenotype of CD4 / CD8 T cells can also express CD95(Fas), CD127(IL7R), and CD27. Addition of low doses of the cytokines IL-7 and IL-15 results in CD45RA int CD45RO intHigh doses of cytokines IL-7 and IL-15 may lead to effector differentiation of the cells, whereas high doses of cytokines IL-7 and IL-15 may lead to the formation of enriched populations of CD4 / CD8 cells with the phenotype.

[0084] In some embodiments, CD45RA int CD45RO int CD4 / CD8 T cells with the phenotype can be genetically modified so that they lack functional CCR5 and / or CXCR4 HIV coreceptors. int CD45RO int Administration of CCR5 and / or CXCR4 gene-edited autologous CD4 / CD8 T cells with the phenotype can result in a sustained increase in CD4+ T cell numbers in the subject, restoration of T cell homeostasis, and a significant reduction in the size of the HIV reservoir.

[0085] Upon transfer or administration to a subject, CD45RA int CD45RO int The enriched population of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells with the phenotype has the ability to persist or survive for a long period in the subject. Persistence can be correlated with the effectiveness of therapeutic T cell transplantation in the treatment of diseases such as HIV infection. The higher the persistence of therapeutic T cells, the more likely the therapeutic regimen is to be effective. Therefore, CD45RA int CD45RO int Long-lived, self-renewing, and multipotent CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells with the phenotype could reduce production costs, promote effector differentiation, and increase the efficacy of treating latent HIV infection in subjects. Furthermore, the frequency of these cells in currently available HIV treatment products can be used as a biomarker to predict the success of interventions.

[0086] In some embodiments, CD45RA int CD45RO intThe enriched population of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells having the phenotype CD45RA int CD45RO int 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 CD45RA phenotype. int CD45RO int Enriched populations with the phenotype CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells may also have an increased ability to engraft in subjects following administration. Specifically, CD45RA int CD45RO int Enriched populations with the phenotype CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells may have an increased ability to engraft in unconditioned recipients.

[0087] The term "engraftment" refers to the ability of transplanted cells to establish in the recipient and survive immediately after transplantation. Therefore, engraftment is assessed shortly after transplantation. For example, engraftment can refer to the number of cells derived from transplanted cells detected at the earliest time point at which transplanted cells or their progeny can be detected in the recipient during the initial in vivo evaluation of an experiment, clinical trial, or therapeutic protocol. In one embodiment, engraftment is assessed 0-12, 0-24, 0-48, or 0-72 hours after transplantation. In another embodiment, engraftment is assessed at about 1, 2, 3, 4, 5, 6, 12, 24, 36, 48, 60, or 72 hours after transplantation. In a preferred embodiment, engraftment is assessed at about 12 hours after transplantation.

[0088] Figure 1 shows that CD45RA can be genetically modified to lack functional CCR5 and / or CXCR4 HIV coreceptors. int CD45RO int1 shows a flow diagram illustrating a method for generating an enriched population of CD4 / CD8 T cells having a phenotype. 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 include any T cell-containing sample from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a 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, T cells can be obtained from the subject with HIV to be treated, i.e., from autologous T cells from the subject to be treated. In certain embodiments, T cells can be obtained from a unit of blood collected from the subject using any number of techniques known to the skilled artisan, such as Ficoll separation. In some embodiments, cells from an individual's circulating blood are 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. 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, cells can be washed with phosphate-buffered saline (PBS). In alternative embodiments, the wash solution can lack calcium, lack magnesium, or lack most or all divalent cations. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS. Alternatively, undesirable components of the apheresis sample can be removed and the cells 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 tissue. In either 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 unique to the negatively selected cells. One method is negative magnetic immunoadhesion or flow cytometric cell sorting and / or selection 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, a monoclonal antibody cocktail typically contains antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0092] For isolation of a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. 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 concentration of cells) to ensure maximum contact between the cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In a further embodiment, a concentration of 125 million or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell proliferation.

[0093] Following isolation of T cells from the biological sample, in step 20, the isolated CD4 / CD8 T cells may be activated and / or expanded by any suitable method known in the art. In embodiments of the invention, T cells are activated and the number of T cells expanded in the presence of one or more non-specific 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 expanded by physically contacting the T cells with one or more non-specific T cell stimuli and / or one or more cytokines. Any one or more non-specific T cell stimuli can be used in the methods of the present invention. Examples of non-specific T cell stimuli include anti-CD3 antibodies and anti-CD28 antibodies. In some embodiments, the non-specific T cell stimuli can be anti-CD3 antibodies and anti-CD28 antibodies bound to beads. Any one or more cytokines can be used in the methods of the present invention. Exemplary cytokines include interleukin (IL)-2, IL-7, IL-21, and IL-15.

[0094] Following activation and / or expansion of the isolated CD4 / CD8 T cells, in step 30, the CD4 / CD8 T cells are assayed for intermediate co-expression of CD45RA and CD45RO (CD45RA), e.g., using flow cytometry. int CD45RO intThe cells may be separated or sorted into an enriched population of CD4 / CD8 T cells characterized by a specific marker (e.g., a specific marker for CD4 / CD8 T cells). The method may include sorting the cells by any suitable method. In some embodiments, the sorting is performed using flow cytometry. Flow cytometry can be performed using any suitable method known in the art. Flow cytometry can use any suitable antibodies and stains. In some embodiments, the flow cytometry is multicolor flow cytometry.

[0095] CD45RA produced by the process described herein int CD45RO int An enriched population of CD4 / C8 T cells with the phenotype CD45RA int CD45RO int In some embodiments, the processes described herein may include CD4 / C8 T cells with CD45RA as the majority cell type. 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 ... The method produces cell cultures and / or cell populations comprising at least about 74%, at least about 73%, at least about 72%, at least about 71%, at least about 70%, at least about 69%, at least about 68%, at least about 67%, at least about 66%, at least about 65%, at least about 64%, at least about 63%, at least about 62%, at least about 61%, at least about 60%, at least about 59%, at least about 58%, at least about 57%, at least about 56%, at least about 55%, at least about 54%, at least about 53%, at least about 52%, at least about 51%, or at least about 50% CD4 / C8 T cells. In a preferred embodiment, the cells of the cell cultures or cell populations comprise human cells.

[0096] CD45RA int CD45RO int The phenotype of long-lived CD4 / CD8 T cells may also be characterized by the expression of other cell surface markers, e.g., CD45RA int CD45RO int The isolated CD4 / CD8 T cells having the phenotype can express at least one of CD95, CD127, or CD27. In other embodiments, CD45RA int CD45RO int CD4 / CD8 T cells with the phenotype can also intermediately express 4-1BB and occasionally express 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 In another embodiment, the isolated T cells may have a CD95+CD127+CD27+ phenotype. int CD45RO int They can have a CD95+CD127+CD27+IL7R+CD44+SCL38A1+IL2RG+CD6+CD5+ phenotype.

[0099] In some embodiments, CD45RA int CD45RO int Before and / or after isolating or sorting CD4 / C8 T cells with the phenotype CD45RA int CD45RO int Isolated CD4-CD8 T cells with the phenotype can be enriched by culturing isolated CD4 / CD8 T cells in cell culture medium containing low amounts of IL-7 and / or IL-15. Activated CD4 / CD8 T cells cultured under low IL-7 / IL-15 conditions (e.g., IL7 / IL15 concentrations less than 10 ng / ml) exhibit a higher CD45RA phenotype than activated CD4 / CD8 T cells cultured under high IL-7 / IL-15 conditions (e.g., IL-7 / IL-15 concentrations greater than 10 ng / ml). int CD45RO int It has been found that it is possible to promote or generate an enriched population of CD4 / C8 T cells with the phenotype.

[0100] In some embodiments, the culture medium can contain IL-7 and / or IL-15 at a concentration of, e.g., 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 less than about 1 ng / ml.

[0101] Using the low IL-7 / IL-15 concentration culture medium described herein, cell populations or cell cultures can be cultured to detect CD45RA int CD45RO int In some embodiments, the phenotypic content of CD4 / C8 T cells can be enriched at least about 2-fold to about 1000-fold compared to untreated cell populations or cell cultures. int CD45RO int CD4 / C8 T cells having the phenotype can be enriched at least about 5 to about 500-fold compared to untreated cell populations or cell cultures. int CD45RO int In yet another embodiment, CD4 / C8 T cells having the phenotype can be enriched at least about 10 to about 200-fold compared to untreated cell populations or cell cultures. int CD45RO int In yet another embodiment, CD4 / C8 T cells having the phenotype can be enriched at least about 20 to about 100-fold compared to untreated cell populations or cell cultures. int CD45RO int CD4 / C8 T cells having the phenotype can be enriched at least about 40 to about 80-fold compared to untreated cell populations or cell cultures. int CD45ROint CD4 / C8 T cells having the phenotype can be enriched at least about 2 to about 20-fold compared to untreated cell populations or cell cultures.

[0102] In some embodiments, once isolated or sorted, the 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. int CD45RO int Addition of TGFβ and / or IL-1β to CD4 / CD8 cells having the phenotype CD45RA prior to administration to a subject int CD45RO int This may lead to maintenance of the phenotype.

[0103] In some embodiments, the method may further include genetically modifying CD4 / CD8 T cells before or after activation and / or isolation so that they lack functional CCR5 and / or CXCR4 HIV coreceptors. When HIV infects human T cells, entry into the cells relies on the association of the T cell receptor CD4 with one of two coreceptors, the chemokine receptor CCR5 or CXCR4. A naturally occurring CCR5 variant ("CCR5-Δ32") in the human population has been identified that appears to be resistant to HIV infection, particularly in the 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, one or both coreceptors can be disrupted to render the cells resistant to the virus (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 the patient to treat HIV.

[0104] In some embodiments, the CD4 / CD8 T cells are CD45RA int CD45RO intPrior to separating the population of CD4 / CD8 T cells having the phenotype from the isolated T cells, the CD4 / CD8 T cells can be genetically modified to lack functional CCR5 and / or CXCR4 HIV coreceptors. In other embodiments, the CD45RA int CD45RO int A population of CD4 / CD8 T cells with the phenotype CD45RA was isolated after separation from isolated T cells. int CD45RO int A population of CD4 / CD8 T cells having the phenotype can be genetically modified to lack functional CCR5 and / or CXCR4 HIV coreceptors.

[0105] In some embodiments, genetic modification or genome editing of CD4 / CD8 T cells can be performed by transduction, transfection, or electroporation. Transduction can be performed by electroporation or transfection with lentivirus, gamma-, alpha-retrovirus, or adenovirus, or nucleic acids (DNA, mRNA, miRNA, antagomir, ODN), proteins, site-specific nucleases (zinc finger nucleases, TALEN, CRISP / R), self-replicating RNA viruses (e.g., equine encephalopathy virus), or integration-deficient 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-stranded DNA breaks, which can then be repaired by either nonhomologous end joining (NHEJ) or homology-dependent repair (HDR), allowing for the insertion or modification of genes using templates homologous to the DNA surrounding the double-stranded break. Traditionally, genome editing is performed by transfecting or transducing cells with RNA or DNA, which then produces proteins and, in the case of the CRISPR-Cas system, guide RNAs required for genome editing.

[0107] For example, double-strand break (DSB) can be created by site-specific nuclease such as zinc finger nuclease (ZFN) or TAL effector domain nuclease (TALEN).See, for example, Urnov et al. (2010) Nature 435(7042):646-51, U.S. Patent Nos. 8,586,526, 6,534,261, 6,599,692, 6,503,717, 6,689,558, 7,067,317, 7,262,054 (the disclosures of which are incorporated by reference in their entirety for all purposes).

[0108] Another nuclease system involves the use of a so-called adaptive immune system found in bacteria and archaea known as the CRISPR / Cas system. CRISPR / Cas systems are found in 40% of bacteria and 90% of archaea, and the complexity of these systems varies. See, for example, U.S. Patent No. 8,697,359. CRISPR loci (clustered regularly interspaced short palindromic repeats) are regions within an organism's genome where short segments of foreign DNA are integrated between short repetitive palindromic sequences. These loci are transcribed, and the resulting RNA transcript ("pre-crRNA") is processed into a 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 contain a Cas endonuclease that processes the pre-crRNA, which, once fully processed into crRNA, assembles into 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: a trans-activating RNA (tracrRNA), complementary to the repeat sequence of pre-crRNA, triggers processing by double-strand-specific RNase III in the presence of Cas9 protein. Cas9 can then cleave target DNA complementary to the mature crRNA; however, 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 the PAM sequence (protospacer adjacent motif). Furthermore, tracrRNA must be present to base pair with the crRNA at its 3' end; this association triggers Cas9 activity.

[0110] The Cas9 protein contains at least two nuclease domains, one similar to the HNH endonuclease and the other similar to the Ruv endonuclease domain. The HNH-type domain appears to be responsible for cleaving the DNA strand complementary to the crRNA, whereas the Ruv domain cleaves the non-complementary strand.

[0111] The requirement for a crRNA-tracrRNA complex can be circumvented by using engineered "single guide RNAs" (sgRNAs) containing the hairpin normally formed by annealing of the crRNA and tracrRNA (see Jinek et al. (2012) Science 337:816 and Cong et al. (2013) Sciencexpress / 10.1126 / science.1231143). In S. pyrogenes, once a double-stranded RNA:DNA heterodimer forms between the Cas-associated RNA and the target DNA, the engineered tracrRNA:crRNA fusion or sgRNA guides Cas9 to cleave the target DNA. This system, which includes the Cas9 protein and an engineered sgRNA containing 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 HIV receptors to prevent HIV infection of 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 in cells to combat 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 HIV receptor expression, CCR5 ribozymes, siRNA sequences targeted to HIV polyproteins, sequences encoding Trim5α restriction factors, sequences encoding APOBEC3G restriction factors, sequences encoding RevM10 proteins, sequences encoding C46, ​​other anti-HIV genes, suicide cassettes, and combinations thereof. Thus, the methods and compositions of the present invention can be used to treat or prevent HIV with a CRISPR / Cas system in which a single guide RNA contains a sequence that targets the CCR5 or CXCR4 gene for integration of a suitable anti-HIV transgene.

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

[0114] In addition to the insertion methods described above, genome editing can also include gene knockout. In the absence of donor nucleic acid, cells with a broken genome utilize the error-prone NHEJ pathway to repair the break. This process often adds or deletes 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 are used in phase I / II trials to create non-functional CCR5 receptors in T cells, thus preventing 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 CCR5 alleles with a CRISPR / Cas system, and a single guide RNA contains a sequence that specifically targets the human CCR5 gene (chr3:46411633-46417697), specifically at or near the exon region (chr3:46414394-46415452).

[0116] One particularly preferred region of the CCR5 gene to target for knockout is the region near the delta-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 region at or near the ATG protein translation start site (chr3:46414347-46414466 or nearby) is also particularly preferred for genomic modifications, such as fusing the C34 peptide to the N-terminus of CCR5 for targeted integration of anti-HIV therapy.

[0117] Similar studies are underway in animal models of CXCR4-dependent HIV, in which CXCR4 is selectively disrupted or disrupted in parallel with CCR5 to prevent HIV infection of T cells (see U.S. Patent Publication No. 2010 / 0291048). Thus, the methods and compositions described herein can be used to disrupt CXCR4 alleles with a CRISPR / Cas system, where the single guide RNA contains sequences that target the human CXCR4 gene (chr2:136871919-136875725), specifically in the region surrounding or near exon 2 (chr2:136872439-136873482) and small exon 1 (chr2:136875616-136875630). One preferred region for targeting the CXCR4 gene for knockout is at or near chr2:136872863-136872982, which is analogous to the delta-32 mutation region of the CCR5 gene. The region at or near chr2:136875540-136875687 near the ATG protein translation start site of exon 1 is particularly preferred, while the region at or near chr2:136873389-136873558 near the splice site of exon 2 is particularly preferred for gene modification, such as fusion of the C34 peptide to the N-terminus of CXCR4 via targeted integration for anti-HIV therapy. Therefore, sgRNAs can be designed to bind to sequences anywhere in the CCR5 or CXCR4 locus, including, but not limited to, sequences from one or more of these preferred target regions.

[0118] Nucleases, polynucleotides encoding these nucleases, donor polynucleotides, and compositions comprising the proteins and / or polynucleotides described herein are administered ex vivo to CD4 / CD8 T cells and / or CD45RA T cells by any suitable means. int CD45RO int The antibody can be delivered to CD4 / CD8 T cells with the phenotype

[0119] Methods of delivering the nucleases described herein are described, for example, in U.S. Patent Nos. 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, the disclosures of all of which are incorporated herein by reference in their entireties.

[0120] The nuclease and / or donor construct described herein can also be delivered using a vector containing one or more sequences encoding CRISPR / Cas systems.Any vector system can be used, including but not limited to, plasmid vector, DNA minicircle, retrovirus vector, lentivirus vector, adenovirus vector, poxvirus vector; herpesvirus vector and adeno-associated virus vector, etc., and combinations thereof.See also U.S. Patent Nos. 6,534,261, 6,607,882, 6,824,978, 6,933,113, 6,979,539, 7,013,219, and 7,163,824, and U.S. Patent Publication No. 2014 / 0335063 (these are incorporated herein by reference in their entirety).In addition, it will be apparent that any of these vectors can contain one or more sequences required for therapy. Thus, when one or more nucleases and donor constructs are introduced into a cell, the nucleases and / or donor polynucleotides can be carried on the same or different vectors. When multiple vectors are used, each vector can contain sequences encoding one or more nucleases and / or donor constructs.

[0121] Conventional viral and non-viral gene transfer methods can be used to introduce nuclease-encoding nucleic acids and donor constructs into cells (e.g., mammalian cells) and target tissues. Non-viral 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 episomal 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); Haddada et al., in Current Topics See Microbiology and Immunology, Doerfler and Bohm (eds.) (1995), and Yu et al., Gene Therapy 1:13-26 (1994).

[0122] Non-viral methods for nucleic acid delivery include electroporation, lipofection, microinjection, particle bombardment, virosomes, liposomes, immunoliposomes, polycation or lipid: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 to deliver nucleic acids.

[0123] Additional exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.), and Copernicus Therapeutics Inc. (see, e.g., U.S. Pat. No. 6,008,336). Lipofection is described, for example, in U.S. Pat. Nos. 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 of Feigner, WO91 / 17424, 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 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 the nucleic acid to be delivered in EnGeneIC delivery vehicles (EDVs). These EDVs are specifically delivered to target tissues using bispecific antibodies, where one arm of the antibody has specificity for the target tissue and the other arm has specificity for the EDV. The antibody carries the EDV to the surface of the target cell, and then endocytosis carries the EDV into the cell. 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 delivery of nucleic acids encoding engineered CRISPR / Cas systems takes advantage of highly evolved processes for targeting viruses to specific cells in the body and transporting the viral payload to the nucleus. Viral vectors can be administered directly to subjects (in vivo) or used to treat cells in vitro, with the modified cells then administered to subjects (ex vivo). Traditional virus-based systems for delivery of CRISPR / Cas systems include, but are not limited to, retroviral, lentiviral, adenoviral, 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 repeats capable of packaging up to 6-10 kb of foreign sequence. The minimal cis-acting LTRs are sufficient for vector replication and packaging, which are then used to integrate therapeutic genes into target cells and provide permanent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., 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 are capable of very high transduction efficiency in many cell types and do not require cell division. High titer and high level expression have been obtained using such vectors. This vector can be produced in large quantities using a relatively simple system. 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, and for in vivo and ex vivo gene therapy procedures (see, for example, 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)). The construction of recombinant AAV vectors has been described in many publications, including U.S. Pat. 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 involving complementation of a defective vector by a gene inserted into a helper cell line to generate the transducing agent.

[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 a gene therapy trial (Blaese et al., Science 270:475-480 (1995)). Transduction efficiencies of over 50% have been observed with MFG-S-packaged vectors (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 the defective, nonpathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from plasmids that retain only the AAV 145 base pair (bp) inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to 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 AAVrhlO, as well as all their variants, can also be used in accordance with the present invention.

[0132] Replication-deficient recombinant adenoviral vectors (Ad) are produced at high titers and readily infect many different cell types. Most adenoviral vectors are engineered so that a transgene replaces the Ad E1a, E1b, and / or E3 genes. The replication-defective vector is then propagated in human 293 cells, supplying the deleted gene function in trans. Ad vectors can transduce multiple tissue types in vivo, including non-dividing, differentiated cells such as those found in liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity. Examples of the use of Ad vectors in clinical trials include polynucleotide therapy for antitumor immunization via intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use of adenoviral 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), Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

[0133] Packaging cells are used to form viral particles capable of infecting host cells. Such cells include 293 cells, which package adenovirus, and psi2 or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are typically generated by producer cell lines that package nucleic acid vectors into viral particles. The vectors typically contain the minimal viral sequences necessary for packaging and subsequent integration into the host (if applicable); other viral sequences are replaced by expression cassettes encoding the proteins to be expressed. Missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically contain only the inverted terminal repeat (ITR) sequences from the AAV genome, which are necessary for packaging and integration into the host genome. Viral DNA is packaged into a cell line that contains a helper plasmid encoding other AAV genes, namely rep and cap, but lacks the ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus facilitates AAV vector replication and expression of AAV genes from the helper plasmid. Due to the lack of ITR sequences, 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 vectors target CD4 / CD8 T cells and / or CD45RA int CD45RO intThe gene editing vector can be delivered ex vivo to CD4 / CD8 T cells with the phenotype, followed by re-implantation of the cells into the patient, usually after selection of cells that have incorporated the vector. Formulations containing gene editing vectors for ex vivo administration can include suspensions in liquids or emulsified liquids. The active ingredient is often mixed with an excipient that is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof. In addition, the formulation can contain small amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, or other agents that enhance the effectiveness of the pharmaceutical composition.

[0135] CD45RA produced by the methods described herein int CD45RO int The selected enriched population of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells with phenotype can be included in a composition, such as a pharmaceutical composition, for treating HIV infection in a subject.The composition can also include a pharmaceutically acceptable carrier.For pharmaceutical compositions, the carrier can be any of those conventionally used for administering cells.Such pharmaceutically acceptable carriers are well known to those skilled in the art and are generally readily available.Preferably, the pharmaceutically acceptable carrier does not have any adverse side effects or toxicity under the conditions of use.

[0136] The composition can be prepared in a unit dosage form for administration to a subject. The amount and timing of administration are left to the discretion of the treating clinician to achieve the desired results. 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 The enriched population of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells having the phenotype CD45RA are formulated for parenteral administration, such as intravenous administration. int CD45RO intA composition comprising an enriched population of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells with the phenotype can be used, for example, to treat HIV in a subject.

[0137] Compositions for administration may be CD45RA provided in a pharmaceutically acceptable carrier, such as an aqueous carrier. int CD45RO int The compositions may contain solutions of enriched populations of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells with the phenotype. 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 auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and auxiliary substances, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The number of cells or CD45RA in these preparations may be varied. int CD45RO int The concentration of the phenotypically CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells can vary widely and will be selected according to the particular dosage form selected and the needs of the subject based primarily on fluid volume, viscosity, body weight, etc. Actual methods for preparing such dosage forms for use in gene therapy, immunotherapy and / or cell therapy will be known or apparent to those skilled in the art.

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

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

[0140] CD45RA int CD45RO int The number of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells with the phenotype also increased with increasing CD45RA int CD45RO intThe presence, nature, and extent of any adverse side effects that may accompany the administration of an enriched population of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells having the phenotype CD45RA T cells will also be determined. Typically, the attending physician will determine the optimal dose of the CD45RA T cells to be used to treat each individual patient, taking into account various factors such as age, weight, general health, diet, sex, route of administration, and the severity of the condition being treated. int CD45RO int One would determine the number of CCR5 and / or CXCR4 gene edited CD4 / CD8 T cells of the invention having the phenotype. By way of example, and not intended to limit the invention, CD45RA int CD45RO int The number of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells with the phenotype is approximately 10 x 10 per infusion. 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 may be a cell.

[0141] CD45RA int CD45RO int It is contemplated that CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells having the phenotype can be used in methods of treating or preventing HIV infection in a subject in need thereof. In this regard, the methods of treating or preventing HIV infection in a subject include administering to a subject a gene encoding a CD45RA T cell as described herein. int CD45RO int The method may include administering to a subject an enriched population of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells, which may have a phenotype, in an amount effective to treat or prevent HIV in the subject.

[0142] In some embodiments, administration of the composition or enriched T cell population to a subject with HIV can promote at least one of a sustained increase in absolute CD4 cell count, restoration of HIV-specific T cell immunity, and substantial decay of the HIV reservoir in the subject. In some embodiments, the subject has been and / or continues to be on antiretroviral therapy.

[0143] CD45RA int CD45RO int The administered CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells having a phenotype can be cells that are allogeneic or autologous to the host or subject. Preferably, the cells are autologous to the subject.

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

[0145] HDAC inhibitors have been demonstrated to induce the transcriptional activation of the HIV-1 promoter. HDAC inhibitors can be any molecule that reduces the activity of histone deacetylase. This includes proteins, peptides, DNA molecules (including antisense), RNA molecules (including iRNA agents and antisense), and small molecules. In some embodiments, HDAC inhibitors are small interfering RNA (siRNA), such as si / shRNA directed against HDAC1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. Non-limiting examples of such HDAC inhibitors are shown 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, HDAC inhibitors may include short chain fatty acids (e.g., sodium butyrate, isovaleric acid, valeric acid, 4-phenylbutyric acid (4-PBA), phenylbutyric acid (PB), propionic acid, butyramide, isobutyramide, phenylacetic acid, 3-bromopropionate, tributyrin, valproic acid (Vpa), valproate, valproic acid hemisodium, 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-carboxycinnamic acid bishydroxamic acid (CBHA), pyroxamide, salicyl bishydroxamic acid, suberoyl bishydroxamic acid (SBHA), azelaic acid bishydroxamic acid (ABHA), azelaic acid-1-hydroxamic acid-9-anilide (AAHA), 6-(3-chlorophenylureido)caproic acid hydroxamic acid (3Cl-UCHA), oxamflatin [(2E)-5-[3-[(phenylsulfonyl)amino]phenyl]-pent-2-en-4-ynohydroxamic acid], A-161906 Can comprise Scriptaid, PXD-101 (Prolifix), LAQ-824, CHAP, MW2796, MW2996; or any of the hydroxamic acids disclosed in U.S. Patent No. 5,369,108, U.S. Patent No. 5,932,616, U.S. Patent No. 5,700,811, U.S. Patent No. 6,087,367 and U.S. Patent No. 6,511,990.In certain embodiments, HDAC inhibitor is SAHA.

[0148] In still other embodiments, HDAC inhibitors may include benzamide derivatives (e.g., CI-994; MS-275 [N-(2-aminophenyl)-4-[N-(pyridin-3-ylmethoxycarbonyl)aminomethyl]benzamide] and 3'-amino derivatives of MS-275.

[0149] In still other 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, apicidin cyclic tetrapeptide [cyclo(NO-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)], apicidin Ia, apicidin Ib, apicidin Ic, apicidin IIa, and apicidin IIb, CHAP, HC toxin cyclic tetrapeptide, WF27082 cyclic tetrapeptide, and chlamydocin.

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

[0151] CD45RA int CD45RO int CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells having the phenotype, alone or in combination with an activator of latent HIV expression described herein, can be administered to a subject latently infected with HIV, e.g., a human latently infected with HIV. The subject can include a subject with a persistent HIV reservoir despite treatment with antiretroviral therapy (e.g., HAART). Thus, in some embodiments, a therapeutically effective amount is a CD45RA gene-edited T cell for significantly reducing the latent HIV reservoir in a latently HIV-infected subject. int CD45RO int The amount of CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells with the phenotype.

[0152] In another embodiment, a therapeutically effective amount of CD45RA int CD45RO int The CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells having the phenotype, and optionally an activator of latent HIV expression, can be administered to a subject in combination with another therapeutic agent, which is useful in the treatment of HIV infection, such as a component used in HAART or an immunotoxin.

[0153] As mentioned above, the CD45RA int CD45RO int The CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells with the phenotype may be combined with one or more additional therapeutic agents useful in the treatment of HIV infection. int CD45RO int It will be understood that the scope of combinations of phenotypically CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells with HIV / AIDS antivirals, immunomodulators, anti-infectives or vaccines is not limited to the following list, but includes, in principle, any combination with any pharmaceutical composition useful in the treatment of AIDS. HIV / AIDS antivirals and other agents will typically be used in these combinations in their conventional dosage ranges and regimens as reported in the art.

[0154] Examples of antiviral medications include (but are not limited to): Antiviral Manufacturer (trade name and / or drug name location) Indication (activity): Abacavir GlaxoSmithKline HIV infection, AIDS, ARC, GW 1592 (ZIAGEN) (nRTI); 1592U89 Abacavir + GlaxoSmithKline HIV infection, AIDS, ARC (nnRTI); Lamivudine + (TRIZIVIR) Zidovudine Acemannan 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 alpha 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 labile alpha abnormality Concepts (Rockville, Md.) Interferon) 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); capravirin Pfizer HIV infection, AIDS, (AG-1549, S-1153) ARC (nnRTI); CI-1012 Warner-Lambert HIV-1 infection Zidofovir Gilead Science CMV retinitis, herpes, papillomavirus Curdlan sulfate AJI Pharma USA HIV infection, cytomegalovirus immunity MedImmune CMV retinitis globin cytovene Syntex Sight-threatening CMV ganciclovir peripheral CMV retinitis delavirdine 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) (combined with AZT / d4T) (nRTI) DPC 681 & DPC 684 DuPont HIV infection, AIDS, ARC (PI) DPC 961 & DPC 083 DuPont HIV infection AIDS, ARC (nnRTRI); Embryne 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 shingles, herpes simplex emtricitabine Triangle Pharmaceuticals HIV infection, AIDS, ARC FTC (COVIRACIL) (nRTI); Emory University 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 beta (Almeda, Calif.); ARC interferon alpha-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 diseases lamivudine; 3TC Glaxo Wellcome HIV infection, AIDS, (EPIVIR) ARC; or with AZT (nRTI); lobucavir 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); Pentafside Trimeris HIV infection, AIDS, ARC T-20 (fusion inhibitor); Peptide T Peninsula Labs AIDS octapeptide (Belmont, California) sequence PRO 542 Progenics HIV infection, AIDS, ARC (attachment inhibitor); PRO 140 Progenics HIV infection, AIDS, ARC (CCR5 coreceptor inhibitor); Trisodium Astra Pharm. Products, CMV retinitis, HIV infection, phosphonoformate Inc. Other CMV infection; 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, in combination with other therapies (nRTI); [PI = protease inhibitor, nnRTI = non-nucleoside reverse transcriptase inhibitor, NRTI = nucleoside reverse transcriptase inhibitor].

[0155] The additional therapeutic agents may be administered individually, sequentially, or in combination with CD45RA int CD45RO int It can be used in combination with CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells having the phenotype, and can be administered to a subject by the same or different routes of administration, or together in the same pharmaceutical formulation.

[0156] According to this embodiment, CD45RA int CD45RO int CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells with phenotype and activators of latent HIV expression can be co-administered with any HAART regimen or its components. The current standard of care with HAART is usually a combination of at least three nucleoside reverse transcriptase inhibitors, often including a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor. CD4+ Subjects with low cell counts or high plasma RNA levels may require more aggressive HAART. + Subjects with relatively normal cell counts and low or undetectable plasma HIV RNA levels over time (i.e., slow or non-progressing) may require less aggressive HAART. Different combinations (or cocktails) of antiretroviral drugs can be used for antiretroviral-naive subjects treated with an initial antiretroviral regimen.

[0157] Thus, in some embodiments, CD45RA int CD45RO int The CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells having the phenotype, and optionally an activator of latent HIV expression, can be co-administered to a subject with a "cocktail" of nucleoside reverse transcriptase inhibitors, non-nucleoside HIV reverse transcriptase inhibitors, and protease inhibitors. For example, CD45RA int CD45RO int CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells with the phenotype and an HDAC inhibitor can be co-administered 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)). int CD45RO int The phenotype-containing CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells, and optionally an activator of latent HIV expression, such as an HDAC inhibitor, can also be co-administered to a subject with a cocktail of one nucleoside reverse transcriptase inhibitor (e.g., stavudine (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 intThe phenotype-containing CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells, and optionally an HDAC inhibitor, can be co-administered to a subject 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] Co-administration in the context of this invention is defined to mean the administration of multiple therapeutic agents in the course of coordinated treatment to achieve an improved clinical outcome. Such co-administration may also be coextensive, i.e., occurring during overlapping periods of time.

[0159] In additional embodiments, the immunotoxin is CD45RA int CD45RO int CCR5 and / or CXCR4 gene-edited CD4 / CD8 T cells with the phenotype can be co-administered to a subject. An example of an immunotoxin is an immunotoxin that targets an HIV protein expressed on the outside of a cell, such as a viral envelope glycoprotein or a portion thereof. The term "immunotoxin" refers to the covalent or non-covalent attachment of a toxin to an antibody, such as an anti-HIV envelope glycoprotein antibody. The toxin can be directly linked to the antibody or indirectly linked, for example, via a linker molecule. The toxin can be selected from the group consisting of ricin-A and abrin-A.

[0160] Activation of latent HIV expression (also referred to as reactivation of latent HIV expression) results in the conversion of latently infected cells to productively infected cells. This transition can be measured by any characteristic of active viral infection, such as production of infectious particles, reverse transcriptase activity, secreted antigens, cell surface antigens, soluble antigens, HIV RNA, and HIV DNA. The methods described herein can optionally include a step of determining or detecting activation of latent HIV expression. In one embodiment, such a method includes determining or detecting mRNA, e.g., 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, traditional immunoassays for HIV proteins (such as gp120 and reverse transcriptase), PAGE, Western blotting, or HPLC, as further described herein or known by those skilled in the art.

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

[0163] In some embodiments, global sequencing and 454 pyrosequencing of HIV-based vector constructs and PCR products described herein can be performed to confirm the production and purity of the autologous virus population. 454 is a simple, efficient, and cost-effective means of obtaining approximate genetic diversity in a sample. In an exemplary embodiment, DNA vectors and plasma RNA are amplified with barcoded primers and then sequenced using 454 JR, obtaining an average of approximately 2,000 reads per amplicon / sample.

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

[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 (the SB-728-0902 clinical trial), we found that this intervention increased CD4+ T cell counts and restored 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, we observed a significant long-term attenuation in the size of the total HIV reservoir in the majority of participants, with a decline of 1 log per million cells in 4 of 9 individuals. 10Although these results were generated using measurements of HIV DNA that do not include the HIV replication-competent reservoir, the highly significant observed reduction in the HIV reservoir contrasts sharply with the very stable levels reported during long-term ART and in recent clinical trials using latent reactivators. These outcomes were not observed in uninfected CD45RA int RO int T SCM In a second study (SB-728-1101 clinical trial), a single injection of these CCR5 gene-edited cells resulted in the production of this novel CD45RA int RO int T SCM We confirmed that a subset was generated and that a higher frequency of such cells was associated with improved control of HIV replication after ART interruption. 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 virus, protecting their progeny from infection.

[0166] material and method The SB-728-0902 clinical trial was a phase 1, uncontrolled, open-label, non-randomized study in chronically HIV-infected individuals treated with ART (ClinicalTrials.gov #NCT01044654). The study was sponsored by Sangamo Therapeutics and conducted at two US centers between December 2009 and April 2014. The primary objective of the study was to evaluate the safety and tolerability of ascending doses of autologous CD4+ enriched T cells (SB-728-T cells) edited in the CCR5 gene by ZFN. Secondary objectives included assessing the effects on CD4+ T cell counts, long-term persistence of CCR5 gene-edited cells, homing to the intestinal mucosa, and HIV viral persistence (HIV RNA and proviral DNA). A total of nine participants were enrolled in three ascending dose cohorts (three participants in each cohort). All participants were followed weekly for the first 4 weeks, then monthly for 1 year, and then enrolled in a 3-year safety study. Participant 1-01 underwent a treatment interruption between months 12 and 31.

[0167] The SB-728-1101 clinical trial was a phase 1, uncontrolled, open-label, non-randomized study in chronically HIV-infected individuals treated with ART (ClinicalTrials.gov#NCT01543152). The 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 increasing doses of cyclophosphamide (CTX) pretreatment to promote CD4+ T-cell expansion after administration of 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 / m on the day before SB-728-T cell infusion. 2(Cohort 4, n=3) received CTX at a dose of 0.1 mg / kg / day (Cohort 4, n=3). Participants then received approximately 10-40 billion SB-728-T cells. All participants were followed weekly for the first 4 weeks, bimonthly 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). Secondary objectives included assessing the effect of SB-728-T cells on plasma HIV-1 RNA levels after ART interruption. During treatment interruption, ART was resumed in participants whose CD4+ T-cell counts fell below 500 cells / µL and / or whose HIV-RNA increased to >100,000 copies / mL on three consecutive weekly measurements. With the exception of one participant who withdrew from the study, all participants completed the 1-year study and were enrolled in a 3-year long-term safety study. One participant (03-003) did not discontinue ART.

[0168] The final clinical protocol, amendments, and consent documents were reviewed and approved by the NIH Recombinant DNA Advisory Committee, as well as the Institutional Review Board and Institutional Biosafety Committee (as appropriate) of each study center. All participants provided written informed consent.

[0169] Registration Criteria SB-728-0902 Study Eligible participants were aged 18 years or older and HIV-infected as documented by ELISA. Participants were aviremic (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 leukapheresis. Key exclusion criteria included a SNP in the CCR5 zinc finger nuclease target region, a current or previous AIDS diagnosis, treatment with maraviroc or immunosuppressants, and hepatitis B or C coinfection.

[0170] SB-728-1101 Study Eligible participants were aged 18 years or older and had HIV infection as documented by ELISA. Participants were aviremic on stable ART with CD4+ T-cell counts >500 / μL, infected with R5-tropic HIV, and willing to discontinue current ART during treatment interruptions. Key exclusion criteria included >40 adenovirus-neutralizing antibodies, SNPs in the CCR5 zinc finger nuclease target region, current or previous AIDS diagnosis, treatment with maraviroc or immunosuppressants, and hepatitis B or C coinfection.

[0171] Cell manufacturing Briefly, participants underwent 10L of leukapheresis to collect, enrich, modify, and expand 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 contains a mixture of gene-edited and unedited cells. Expression of the CCR5-specific ZFN induces double-strand breaks in the cells' DNA, which are repaired by the cellular machinery, leading to random sequence insertions or deletions (indels) in approximately 25% of transduced cells. These indels disrupt the CCR5 coding sequence, resulting in frameshift mutations and the 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. Consequently, time points were grouped into early (14–28 days), mid-term (4–7 months or 9–10 months), late (11–12 months), and long-term (2–3 or 3–4 years) time points after infusion. Baseline samples included cryopreserved PBMCs from the first leukapheresis (2–3 months before infusion) and a small blood draw 1–2 weeks before infusion. PBMCs from participants 1-01, 1-02, and 1-03 were not cryopreserved until 6 or 8 months after infusion. Most participants agreed to large-volume blood draws (n = 9, 2–3 years) and / or leukapheresis (n = 7, 3–4 years) during the long-term follow-up period to allow for assays requiring large numbers 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 for all participants.

[0173] SB-728-1101 Clinical measurements (CD4 and CD8 counts, viral load (VL), and pentamer replication markers) were performed at all time points. The availability of cryopreserved PBMCs at baseline and before 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 the first leukapheresis (2–3 months prior to infusion) and a small blood draw 1–2 weeks prior to infusion. Manufacturing samples (SB-728-T product) were also available for participants in cohorts 3–5.

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

[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 to disrupt the CCR5 locus. A PCR-based assay was developed to measure the gain of unique copies of a five-nucleotide (pentamer) DNA sequence, CTGAT, at the ZFN cleavage site in approximately 25% of gene-edited alleles. Genomic DNA (gDNA) was extracted from PBMCs using a commercially available kit (Masterpure DNA Purification Kit, Epicentre, Madison, WI). Standard PCR was performed with 5 μg of gDNA to amplify a 1.1 kb region containing the CCR5 gene modification. This 1.1 kb amplicon was then evaluated in two independent qPCR runs: one specific for the pentamer-copy-CCR5 gene-edited allele (by using primers containing the pentamer copy), and the second amplifying all CCR5 alleles. The ratio of templates specific for the pentamer copy and the total number of CCR5 alleles yields pentamer copies per million PBMCs. The assay was performed on 10 5 With a sensitivity of one CCR5 gene-edited allele per total CCR5 alleles, the frequency of CCR5 gene-edited cells in PBMCs was estimated by multiplying the frequency of pentamer-replicating gene-edited cells by 4.

[0176] Quantification of CCR5 gene modification in SB-728-T products using Cel-I Cel-I nuclease specifically cleaves DNA duplexes at sites of distortion created by either bulges or mismatches in the double-helical DNA structure. We employed a protocol using this enzyme to quantify minor indels, which are typically induced by ZFN-mediated gene modification. Briefly, the genomic region of interest (CCR5) is PCR-amplified, the PCR products are denatured, and then reannealed to allow the wild-type and non-homologous end-joining-edited alleles to reanneal together to create a heteroduplex. The reannealed PCR products are then digested with Cel-I nuclease, which cleaves the PCR-amplified DNA at the sites of mismatches. The level of ZFN-mediated gene modification can then be quantified by determining the ratio of the uncut parent fragment to the two lower-mobility cleavage products.

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

[0178] CCR5 gene-edited CD45RA after SB-728-T infusion int RO int T SCM Cell Tracking Using sequencing of CCR5 ZFN-mediated mutations in sorted CD4+ T cell subsets, we demonstrate the ability of CD45RA to differentiate after infusion. int RO int T SCM Cell differentiation was tracked. First, wild-type CCR5 (amplicon) and sequences detected in only one of the sequenced samples were excluded from further analysis (approximately 80% of unique CCR5 sequences). Then, for each donor, the CD45RA amplicon of the SB-728-T product was analyzed. int RO int T SCMWe identified sequences expressed exclusively in CD4+ T cell memory subsets in 3-4 year samples (SB-728-0902) or 6 and 22 week samples (SB-728-1101) and then analyzed their distribution in CD4+ T cell memory subsets.

[0179] Estimated spread of SB-728-T after infusion The level of CCR5 gene-edited alleles remaining in participants relative to the amount of CCR5 gene-edited cells infused can be estimated using measurements of CCR5 modification by pentamer replication markers and CD4+ cell counts assuming: 1) blood volume is 4.7 liters; 2) approximately 2.5% of all CD4+ T cells are peripherally resident. 49 and 3) the distribution of SB-728-T products is similar to that of endogenous CD4+ T cells (the levels of CCR5 modification on CD4+ T cells from the sigmoid and inguinal nodes are similar to those in the periphery, Figure 8E).

number

[0180] Cellular phenotyping of SB-728-T products in baseline and post-SB-728-T infusion samples Analysis and evaluation of co-inhibitory receptors on CD4+ and CD8+ subsets were performed by T before fixation with 2% FA (Sigma Aldrich) for 15 min at 22°C. SCM One million thawed PBMC surfaces were stained for 30 minutes at 4°C with either the T cell panel or the negative regulator 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 an aqueous fluorescent 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 regulator 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 viable cells were acquired within 24 hours using a BD LSR-II and analyzed using FlowJo version 9.

[0181] Cell sorting For quantification of pentameric replicator and integrated DNA levels within CD4+ T cell subsets, CD4+ T cells were first isolated from PBMCs by negative magnetic selection (StemCell) and then probed for 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 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), CD19 BV Cells were surface stained with 510 (clone H1B19) (Biolegend), CCR7 FITC (clone 150503) (R&D), and aqueous fluorescently reactive dyes (Invitrogen). Up to 200,000 total CD4+ T cells and CD4+ T cell subsets were then sorted using a FACSAria (Becton Dickinson) and stored as dry pellets at -80°C until analysis. For gene array analysis of immune subsets, 10,000 sorted cells were collected directly into 1.5 mL RNAse-free Eppendorf tubes containing 500 μL of RLT buffer with 1% β-mercaptoethanol and stored at -80°C until analysis.

[0182] HIV DNA in PBMCs, total and sorted CD4± T cell subsets Total HIV DNA in PBMCs was measured by droplet digital polymerase chain reaction. Briefly, genomic DNA (gDNA) was extracted from PBMCs using a commercially available kit (Masterpure DNA Purification Kit, Epicentre, Madison, WI). 2 μg of gDNA was digested with the 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™ 2x 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 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 s), 94°C (30 s) / 60°C (60 s), 98°C (600 s)). 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 HIV gag concentrations to RPP30 concentrations. Integrated DNA was measured as previously described in purified CD4+ T cells from SB-728-0902 participants at baseline, SB-728-T product, and year 2–3 samples (n = 9), and in sorted CD4+ T cell subsets in SB-728-T product and year 3–4 samples (n = 8). Integrated DNA was also measured in purified CD4+ T cells and sorted CD4+ T cell subsets from SB-728-1101 Cohorts 3-5 participants in baseline, week 2-6, and week 14-22 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 was determined using a cell-based transduction assay in which HIV env protein sequences were amplified from PBMC samples, subcloned into libraries, packaged into lentiviral vectors, and evaluated using coreceptor-restricted cell lines.

[0184] Intracellular cytokine staining Thawed PBMCs were allowed to rest for 12 hours, and then 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), staphylococcal enterotoxin B (SEB, 1 μg / mL), or complete medium (mock). Cells were then 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 reactive dyes (Invitrogen), permeabilized with 0.05% saponin, and then stained with IL-2 PerCP-Cy5.5 (clone MQ1-17H12), IFNγ APC (clone B27), and TNFα Alexa Fluor 488 (clone MAB11) (BD Biosciences). Cells were stained intracellularly with BD Biosciences and then fixed with 2% formaldehyde. Cells were acquired within 24 hours using a BD LSR-II. At least 500,000 raw events were acquired. Cells were analyzed using FlowJo version 9, and the distribution of polyfunctional CD8+ T subsets 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 rearranged TCR CDR3 sequences by multiplex PCR to explore all Vβ and Jβ combinations from isolated genomic DNA, and then uses high-throughput sequencing technology to sequence the TCR CDR3 chains and determine the composition of various T cell clones within each sample. TCR diversity was assessed using the Shannon entropy index, which describes both the number of unique clones (richness) and the distribution of clones (uniformity) of TCR Vβ CDR3 sequences present in each sample. A larger Shannon entropy index reflects a more diverse distribution of TCR Vβ CDR3 sequences.

[0186] Gene microarray and analysis Selected CD4+ or CD8+ subsets were sorted into RLT buffer as described above. Specifically, CD4+ T CM , CD4+ T TM , CD4+ T EM CD4+ total memory cells and CD8+ total memory cells were sorted at baseline and month 12. Additionally, CD4+ memory subsets were also sorted at years 3 and 4, and CD45RA int RO int T SCM , T CM , and T EMThe cells were sorted and then lysed for RNA extraction according to the manufacturer's instructions (Qiagen, Valencia, CA). In vitro transcription was performed following a T7 oligo(dT)-primed reverse transcription reaction. These products underwent a second round of amplification (MessageAmp II aRNA Amplification Kit by Life Technologies) to generate biotin-labeled aRNA, which was hybridized to Illumina Human HT-12 Version 4 Expression BeadChips according to the manufacturer's instructions and quantified using the Illumina iScan system.

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

[0188] Gene set enrichment analysis (GSEA) was used to identify enriched biological pathways regulated in T memory cells after infusion (Figure 4). GSEA is a statistical method that determines whether members of a particular gene set occur preferentially at the top or bottom of a ranked gene list, in which genes are ranked by the strength of their association with the outcome of interest. More specifically, GSEA calculates an enrichment score (NES), which reflects the degree to which a set of genes is overrepresented among differentially expressed genes. The significance of the observed NES is obtained by a permutation test, and the gene list is re-sorted to determine how frequently the observed NES occurs by chance. A state-of-the-art analysis was performed to examine the specific genes in the gene set that most contributed to the enrichment. We used the pre-ranked gene list option in GSEA and tested for enrichment in curated gene sets and custom gene sets in MSigDB (http: / / software.broadinstitute.org / gsea / msigdb / ) to test for enrichment of Treg and STAT3 pathways in our data. Gene sets with a false discovery rate (FDR) greater than 25% and a nominal P value greater than 0.05 were discarded.

[0189] Using the GSEA described above along with the Fisher composite test approach, CD4+ CD45RA int RO int T SCM Against CD4+ T CM Comparison of CD4+ CD45RA int RO int T SCM Against CD4+ T EMWe identified pathways that were enriched in the comparison of T EM and T CM Compared with both CD45RA int RO int T SCM The selected pathways that were significantly enriched in genes induced or repressed in the IL-16 / ...

[0190] Use a pie chart to visualize CD4+ T CM , T EM and T TM The top enriched pathways increased or decreased at 12 months compared to baseline in the CD8+ total memory subset (Figure 4B, C). To assess whether the gene expression profile of CD4+ memory T cell subsets at 12 months post-infusion mimicked that of immune responders (IR), we performed GSEA analysis on an independent cohort (Cleveland Immunocompromised - CLIF- cohort) of HIV-infected IR (n = 20, CD4+ count > 500 cells / µL) and immunological non-responders (INR, n = 21, CD4+ count < 350 cells / µL) participants. All pathways (5 / 5) upregulated in CD4+ memory T cells at 12 months post-SB-728-T product infusion were enriched in IR compared to INR (Figure 4E, F). These pathways were associated with active metabolism (MYC, OX / PHOS) and proliferation (DNA repair).

[0191] CD4+ T at 12 months compared with baseline CMTo examine the effects of SB-728-T on inflammation and immune activation in CD8+ total memory cells, we first performed a 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 below 0.05. Differentially expressed genes induced by type I interferon (IFN-I) were identified using the Interferon Database (http: / / interferome.its.monash.edu.au / interferome / home.jspx). Selected genes (up- or down-regulated at 12 months compared to baseline) from each comparison were submitted 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 6 Frequency of total HIV DNA copies per PBMC and CD45RA at 3-4 years int RO int T SCM CD45RA in 3-4 year samples correlated with number int RO int T SCM We identified genes expressed by CD45RA. int RO int T SCM Linear models were fitted (using R) between gene expression levels during HIV infection and these outcomes as continuous variables, and pathways were associated positively or negatively with both readouts using GSEA. Pathways regulated in a similar manner, as well as pathways regulated in different directions, are depicted in Figure 4g. HIV reservoir size, as measured by total HIV DNA in years 2–4, was positively correlated with HIV reservoir size, as measured by CD45RA in years 3–4. int RO int T SCMThe top pathways negatively correlated with counts were highlighted by plotting normalized enrichment scores (Figure 4H, I). This analysis was performed in six of the seven participants. int RO int T SCM Participant 1-02, who had low cell engraftment, 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 injection over time (days) (10 6 The HIV risk (measured as total HIV DNA per PBMC) was modeled using a mixed-effects linear regression model with a random intercept, as implemented in the function lmer from the R package lme4. 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 each individual's HIV reservoir, 10 6 The frequency of total HIV DNA per PBMC was fitted to each individual using a linear regression model using 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 by the model fit intercept.

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

[0196] Statistical Analysis of Total HIV DNA Decay After SB-728-T Product Infusion in SB-728-0902 To explain the post-infusion HIV DNA decay observed in six participants, 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, which estimates parameters using a nonlinear mixed-effects model approach. Using both an individual approach and a population approach (comprising all six patients), we estimated a biphasic exponential decay using the equation Y = a + b1 * exp(-r1 * time) + b2 * exp(-r2 * time) (Equation 1). Monolix implements a stochastic approximation of the expectation-maximization algorithm using a Markov chain Monte Carlo (MCMC) iterative algorithm. The MCMC iterative method uses the Metropolis-Hastings approach. To perform the fitting, we considered different distributions for each of the biphasic decay model equations. The two decay rates r1 and r2 took values ​​between (0 and 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, were estimated using a lognormal distribution, taking only positive values. To ensure model convergence, 2000 Monte Carlo iterations were used in the simulation step. The shape of each Monolix fit was also confirmed using the nlsLM package in R, a nonlinear least-squares estimation method, for the same biphasic decay function.

[0197] To estimate the time at which the fast and slow phases of HIV DNA began to dominate, the slopes associated with each of the fast and slow phases were plotted using the following equation: 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 slopes (Equations 2 and 3) indicates the time when the slow phase begins to dominate over the fast decay phase. Similarly, the intersection of the slow phase (Equation 3) with the plateau line indicates the time when the slow decay phase ends and HIV DNA reaches the new frequency level estimated by the plateau. Calculations for each of the six patients were performed using MATLAB.2016.

[0199] Similarly, to estimate the contribution of both the fast and slow phases to total HIV DNA decay, the following equation was used: Percent_Fast: Φ=b1 / (Y0-Plateau)*0.01, Y0=a+b1+b2(HIV DNA copies at t=0 day) Percent_Slow: Y=100-Φ

[0200] Model Selection and Diagnostics Compared to the results of the population approach, the individual approach yielded better fits. Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) were used to assess the differences between the R and Monolix fits, respectively, and the individual vs. population fit results. Small AIC and BIC indicate the most parsimonious model, which was better achieved using the Monolix individual approach.

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

number

[0202] Using the individual fitting approach in Monolix R2018, we fitted ODE models to both CCR5 gene-edited and non-gene-edited CD4+ T cell memory subset counts for five patients with extended ATI interruption periods (weeks 6 to 12 months). Due to the small sample size, we employed the individual fitting routine to parameterize the model parameters, as described in the previous section (Statistical Analysis of Total HIV DNA Decay After SB-728-T Product Infusion in SB-728-0902). We assumed a log-normal distribution with positive values ​​for all model parameters.

[0203] Global sensitivity analysis To determine which parameters most affect cell population size, a sensitivity analysis test was conducted using Latin hypercube sampling (LHS) and partial rank correlation coefficients (PRCC) to measure the linear relationship between model parameters and model output. This test allows for simultaneous investigation of the sensitivity between multiple parameters and model output. To account for uncertainty in the parameter distribution, model parameters were varied using a uniform distribution, where the maximum and minimum values ​​were taken from five individual fits obtained for each subject. A monotonic relationship between model parameters and output 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 infused cells in SB-728-0902 HIV DNA decay after infusion in participants in the SB-728-0902 study due to dilution with the amount of infused cells can be estimated using CCR5 gene editing with a pentamer replication marker and measurement of CCR5 gene editing in the SB-728-T product using Cel-I nuclease, assuming 1) one gene-edited allele represents one gene-edited cell, 2) CD4+ T cells from the SB-728-T product do not contain cells containing HIV DNA, and 3) unedited cells persist similarly to CCR5 gene-edited cells after infusion (participants continued on 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 infused 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 HIV DNA decay due to infused cells = baseline frequency of cells containing HIV DNA * frequency of infused cells in PBMCs at each time point / 100

[0208] Estimated frequency of HIV DNA attributable to infused cells = baseline frequency of cells containing HIV DNA - estimated decay of HIV DNA by infused cells at each time point

[0209] outliers Participant 1-02 had elevated anti-adenovirus titers, and demonstrated increased engraftment levels of CCR5 gene-edited cells and CD45RA, a population highly enriched in gene-edited cells. int RO int T SCM(The SB-728-T product is derived from transduction with a recombinant Ad5 / F35 adenoviral vector encoding a CCR5-targeting ZFN) and therefore may prevent the persistence of CCR5 gene-edited cells and CD45RA int RO int T SCM subsets were excluded from selective analyses focused on correlating proliferation with HIV reservoir decay (e.g., Figures 1c–d, 2d–e, and 3g–i).

[0210] result A single SB-728-T injection resulted in a sustained reduction in HIV reservoir size, which correlated with the proliferation and persistence of CCR5 gene-edited cells. Clinical trial SB-728-0902 evaluated nine HIV-infected adults on long-term ART who had failed to increase their CD4+ T-cell counts to levels above 500 cells / µL. At baseline, participants had been on effective ART for 7 to 22 years and had a mean CD4+ T-cell count of 363 cells / µL. CD4+ T-cell counts were inversely correlated with the level of integrated HIV DNA (referred to here as the HIV reservoir) at the 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 ratios) increased within 7 days after infusion, as expected (see online discussion, Figures 10A and 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 after infusion (median 2.4-fold proliferation at 21 days, Figure 10C) and was associated with increased CD4+ T cell counts. CCR5 gene-edited CD4+ T cells were detected in PBMCs for up to 4 years (average 0.8% of marked PBMCs and average 2.7% of marked CD4+ T cells) and in rectal biopsies and lymph nodes for up to 12 months (last measured time point) (Figures 10D and 10E).

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

[0213] Interestingly, CD4+ T cells from the SB-728-T product had a significantly lower frequency of latently infected cells than CD4+ T cells from baseline (P = 0.004, Figure 16A). A two-phase decay model was used to determine whether the persistence of infused cells containing low levels of integrated HIV DNA contributed solely to decay of the HIV reservoir through dilution during peak proliferation of infused cells. The slope of HIV DNA decay was greatest during the first 1–15 days of infusion, during which a mean decline of 30.47% (95% CI, 9.664–51.28) was observed. Thereafter, HIV DNA levels continued to decline at a slower rate (95% CI, 56–365) with a half-life of 211 days. This slower decay phase accounted for the majority of 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). We found that the observed frequency of cells containing HIV DNA was lower than that estimated by dilution alone after approximately 100 days, demonstrating that dilution alone cannot explain the long-term decline in the frequency of HIV-infected cells. Our analysis suggests that the persistence of infused cells leads to HIV attenuation through mechanisms that may include restoration of T cell homeostasis and / or replenishment of the CD4+ T cell pool with 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 reconstitution, we performed a longitudinal analysis of the distribution of CD4+ T cell subsets after infusion. Our study demonstrated that CD45RA and CD45RO intermediate levels (CD45RA) were associated with a significant increase in CD4+ T cell reconstitution. int RO int The results showed a specific increase in T cells expressing CD45RA (referred to as "CD45RA") (Figure 3A). int RO int Cells present in the SB-728-T product were highly enriched in the CCR5 gene-edited allele, with significant increases in absolute numbers at all time points analyzed after infusion. Importantly, CD45RA int RO intThe changes in cell numbers were significantly correlated with the long-term increase in CD4+ T cell numbers, but not with changes in other memory subsets (Table 1). int RO int and CD45RA + RO - T in the subset SCM The frequency of CD45RA (a marker expressed in CD45RA) positively correlated with proliferation of CCR5 gene-edited cells. int RO int CD95+ cells (CD45RA int RO int T SCM ) and CD45RA + RO - CD95+ cells (CD45RA + T SCM These are specifically enriched in the central memory (T CM ) or transitional memory (T TM ) cells compared with CD45RA int RO int T SCM The levels were approximately 14-fold and 21-fold higher than those in the control group (Figure 3B). Sequencing of the CCR5 DNA mutations driven by gene editing revealed that the T CM 2.5% to 16.7% in T TM 1% to 16.7% in T EM CD45RA compared with a range of 0.7% to 2.59% in int RO int T SCM We confirmed long-term enrichment of CCR5 gene-edited alleles in patients with SB-728-T (range 14.4%-37.7%). Notably, these mutational variations were significantly higher than those observed in patients with CD45RA gene-edited alleles between the SB-728-T product and the 3-4 year timepoints. int RO int T SCM These results suggest that these cells most likely represent a long-lived memory subset that contributes to the long-term polyclonal persistence of CCR5 gene-edited cells.int RO int CD95- or CD45RA + T SCM CD45RA, but not cell persistence int RO int T SCM The persistence of CCR5 mutations was significantly correlated with an increase in total CD4+ T cell counts (Table 1). Collectively, these results suggest that SB-728-T infusion induces de novo T cell proliferation, which is associated with long-term persistence of CCR5 mutations and CD4+ T cell reconstitution. SCM We show that this leads to similar subsets. [Table 1]

[0215] T 3-4 years after injection EM The presence of CCR5 gene mutations in short-lived memory cells such as T SCM and T CM CCR5 gene-edited cells in long-lived memory cells such as T EM and differentiated into CCR5 gene-edited T cells for 3-4 years after injection. EM These findings suggest that CD45RA contributes to the maintenance of a small subset of cells. 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 CD4+ T cell subsets in 3-4 year samples. int RO int T SCM We found that CD45RA cells had significantly lower levels of integrated HIV DNA (1.99 log10, 95% CI: 3-4 years) compared with other memory subsets. int RO int T SCM 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 (2.28-3.45, P=0.023). int RO int T SCM Although only 5.3% of cells contributed to CD4+ T cells in the 3-4 year samples (Fig. 3c), other memory subsets contributed significantly higher cell frequencies to the pool of cells carrying HIV tot DNA [T CM 45.5% (P = 0.0078) for T TM 16.5% for (P = 0.0156), and T EM 29.6% (P = 0.0156)].

[0216] CD45RA carrying low levels of integrated HIV DNA int RO int T SCM Differentiation of cells into other memory subsets could 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 frequency of PBMCs harboring total HIV DNA after infusion and to predict the CD45RA int RO int T SCM Cell count, CD45RA int RO int T SCM The frequency of pentamer replication in cells, as well as CD45RA int RO int T SCM and T EM We included the number of mutations shared between individuals as a possible independent variable. Our analysis demonstrated that greater decay in the HIV reservoir after infusion was associated with higher CD45RA levels at 3–4 years. int RO int T SCM cell count (P=0.0018), CD45RA at 3–4 years int RO int T SCM a higher frequency of pentamer replication in CD45RA (P = 0.005), and int RO int T SCM Frequency of pentamer duplication in T EMThe frequency of pentamer duplication in the 1000-kDa population was best predicted by a lower ratio (P = 0.0014) (adjusted R 2 = 0.99, F test: P = 0.0008, Figure 3D). int ROintT SCM Long-term persistence of cell counts and T EM We demonstrated that maintaining a subset of CCR5 gene-edited cells within the population is important for reducing the HIV reservoir, and demonstrated that CD45RA int RO int T SCM This suggests that the cells differentiate into a pool of more differentiated memory cells that can be replenished with a detectable proportion of cells carrying the CCR5 gene-edited allele that can withstand infection.

[0217] CD45RA int RO int T SCM express genes associated with quiescence and self-renewal and can differentiate into other memory subsets. CD45RA int RO int T SCM Our results demonstrating their impact on the persistence of CD4+ T cells and long-term CD4+ T cell reconstitution suggested that these cells express genes and pathways that confer long-term persistence. Transcriptional analysis of sorted CD4+ T cell subsets was performed on samples from 3-4 years post-infusion. Multidimensional scaling of gene expression changes revealed that CD45RA int RO int T SCM and CD45RA + T SCM And more than that, T CM and T EM In addition, there was a greater difference between CD45RA + T SCM T EM Compared to T CM and CD45RA + T SCM A larger number of differentially expressed genes (DEGs) were found when compared with CD45RA (5022 vs. 2943, vs. 2136). Gene set enrichment analysis (GSEA) revealed that CD45RAint RO int T SCM We showed that cells were enriched in genes involved in stemness (such as the Notch signaling pathway, which is required for Wnt-mediated maintenance of undifferentiated HSCs) and metabolic pathways that contribute to cellular persistence, such as fatty acid oxidation, oxidative phosphorylation, and pyruvate metabolism. CM and T EM 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 cells are more quiescent than other memory subsets.

[0218] CD45RA differentiates into other memory subsets int RO int T SCM To assess the efficacy of the SB-728-T product, we sequenced CCR5 ZFN-mediated mutations in selected CD4+ T cell subsets and identified CD45RA mutations in the SB-728-T product. int RO int T SCM We identified cell-specific sequences (n=3,881). Analysis of the distribution of these sequences 3–4 years post-infusion in various CD4+ T cell memory subsets (Figure 4B) revealed that CD45RA int RO int T SCM Unique mutations lead to the short-lived T EM The results showed that CD45RA was detected in all memory T cell subsets, including 0.49% (95% CI: 0%-1.36%). int RO int T SCM We demonstrate that CD45RA cells are able to differentiate into and replenish a pool of more differentiated memory cells compared to other memory subsets. int RO int T SCMTo further characterize the differentiation state of CD4+ CD45RA cells, we examined their polyfunctional responses after stimulation with anti-CD3 / 28 coated beads (Figure 4c), SEB, or PMA / ionomycin by flow cytometry. int RO int T SCM The cells, T CM T TM , and T EM CD45RA int RO int T SCM The undifferentiated state of the cells was confirmed by analyzing the expression levels of transcription factors associated with Th1 (T-bet and Eomes), Th2 (GATA-3), and Th17 (RORgt) lineage commitment: naive cells and CD45RA. + T SCM Similarly, CD45RA int RO int T SCM The cells did not express Th-specific transcription factors (Fig. 3d). Further analysis of immune checkpoint markers revealed that CD45RA int RO int CD95 + The cells are T TM and T EM showed significantly lower levels of PD-1, TIGIT, and SLAM than CD45RA int RO int CD95 + These results suggest that CD45RA cells are less likely to be expelled than other memory subsets (Figure 28B). int RO int T SCM exhibit stem cell properties, including longevity and pluripotency, and T CM , T EM and T EM This indicates that they are more differentiated progenitor cells than memory cells.

[0219] Next, CD45RA int RO int T SCM and CD45RA + TSCM We compared the transcriptomes of the cells to investigate differences in gene expression and specifically pathways involved in self-renewal, including the Wnt signaling cascade, a pathway implemented in maintaining the "stem-like" phenotype of T cells (Figure 4F). Multidimensional scaling of gene expression variance revealed that the previously characterized CD45RA + CD95 + T memory stem cells (T SCM ) but not the CD45RA described in this study. int RO int T SCM These gene sets were transcriptionally distinct from the IL-16 population (Figure 4E). A closer look at the combined leading-edge genes of these gene sets revealed up-regulation of the expression of several genes important for maintaining stemness. These included Wnt factors and their receptors (Frizzled-FZD), which, as noted above, are known to initiate the stemness maintenance cascade (Figure 4G). This was coupled with downstream signaling cascades including DVL, β-catenin, and TCF7 genes, which are known to prevent effector T cell differentiation. Further downstream mechanisms of stemness, defined by up-regulation of SOX genes, were also observed (Figure 4G). Interestingly, CD45RA int RO int T SCM Those CD45RA + T SCM When compared to their counterparts, they were enriched in pro-inflammatory (MAPK, Jun, NFAT) and apoptotic (PSM) gene sets (Figure 4G). The enhanced pro-inflammatory signature in this subset may indicate an increased ability to differentiate into more "effector"-like cells. Interestingly, a combination of leading-edge genes associated with the Wnt signaling cascade positively correlated with long-term increases in CD4+ T cell numbers and negatively correlated with reduced HIV reservoirs after infusion.

[0220] Taken together, these results suggest that CD45RA int RO int T SCMA novel and distinct T cell phenotype with characteristics of long-lived, undifferentiated memory cells SCM Make sure you configure a subset.

[0221] CCR5 gene-edited CD45RA int RO int T SCM The frequency of SB-728-T correlates with viral load control in participants who underwent a 6-week treatment interruption after SB-728-T infusion. Next, CD45RA int RO int T SCM We evaluated the impact of infusion of CCR5 gene-edited CD4+ T cells containing SB-728-T on viremia control upon ART treatment discontinuation. 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 interruption (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 historical pre-ART viral load set points (P=0.0067) (Figure 5A), indicating that infusion of the SB-728-T product may have led to transient, but incomplete, viremia control in the majority of participants. Prolonged ATI in six individuals demonstrated viral load measurements of less than 10,000 copies / mL and CD4+ T-cell counts above 500 cells / µL at week 22, and subsequently spent 0.5–2 years on ATI. The viral loads at 12 months for the five participants who were currently undergoing ATI ranged from 130 to 16,000 copies / mL. One of these individuals (01-060) had a protective human leukocyte antigen (HLA) allele, HLA-B57.

[0222] Coincidentally, the significant reduction in viral load at week 22 compared to historical pre-ART viral load set points significantly correlated with a larger change in CD4+ T cell counts at peak cell proliferation (Figure 5B) and a higher frequency of CCR5 gene-edited alleles before ATI (week 6) (Figure 5C). These results highlight the association between CCR5 gene-edited cell proliferation and viral load control during SB-728-T infusion, which was also shown to be enriched for CCR5 gene mutations in the 1101 study (Figures 5d, e), suggesting the role of T in reducing viral load after ATI. SCM Correlation analysis between viral load and CD4+ T cell subset counts showed that higher CD45RA before ATI (week 6) int RO int T SCM and CD45RA + RO - T SCM Cell count (especially CCR5 gene-edited T cells) SCM cell count) correlated with a significant reduction in viral load at week 22 compared with historical viral load set points (Fig. 5F,G).

[0223] Next, we investigated the functional responses of HIV-specific CD8+ T cells, which have previously been shown to play an important role in controlling viral replication (REFS) after ATI, and compared the peak frequency of cytokine (IFN-γ, TNF-α, IL-2) production by HIV-specific CD8+ T cell subsets after ATI, as well as CD45RA expression before ATI (week 6). int RO int T SCM We constructed a multivariate regression model to predict change in viral load at week 22 compared to historical pre-ART viral load set points using the number of IL-2-producing CD8+ T cells. Our analysis demonstrated that reduction in viral load at week 22 compared to historical pre-ART set points was associated with a significant association with IL-2-producing CD8+ T cells. TM Higher CD45RA cells were detected before ATI, along with a peak frequency of int RO int T SCM These results suggest that the decay of the HIV reservoir is due to the CD45RA int ROint T SCM cells (p=0.05) and IL-2-producing CD8+ T cells TM demonstrates that it is significantly and negatively associated with proliferation of cells (P=0.02).

[0224] CCR5 gene-edited T EM The frequency of SB-728-T infusions correlated with viral load control in participants who underwent treatment interruption during the 6-week period. CCR5 gene-edited CD45RA int RO int T SCM To test the hypothesis that CD45RA cells contribute to viral load control through differentiation into other memory subsets during ATI, we int RO int T SCM We first identified product-specific CCR5 mutations and followed their persistence in other memory cells after infusion and ATI. Our results demonstrate that CD45RA int RO int T SCM We demonstrate that product-specific CCR5 mutations were detected in all memory subsets CD4+ at weeks 6 and 22 (frequency counts added, Figure 6A), demonstrating that the CCR5 gene-edited CD45RA int RO int T SCM Highlight the ability of cells to differentiate.

[0225] To investigate the impact of long-term viral replication on the persistence and differentiation capacity of CCR5 gene-edited CD4+ T cell memory subsets, we next used an ordinary differential equation model to model the homeostasis of CCR5 gene-edited and unedited CD4+ T cell subsets during viremia (weeks 6–12 months) in five individuals whose ATI extended beyond month 12. Using individual fitting routines in Monolix, we obtained the death (γ), proliferation (ρ), and transition (φ) rates (cells / day) for each CCR5 gene-edited and unedited CD4+ T cell subset. We used the 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-fold lower compared to that of their unedited counterparts (Figure 6B). + 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 sensitivity analysis tests in Matlab to identify the relationship between parameters and cell number, we found that the model parameters for transition were significantly higher in the CCR5 gene-edited CD45RA after ATI. + T SCM , CD45RA int RO int T SCM , and T CM We observed that the CCR5 mutation had a significant negative correlation with cell count (Figure 6B). Collectively, these results suggest that the presence of the CCR5 mutation confers a protective effect on the CD4+ early memory subset, and that the loss of these cells over time is much more likely due to differentiation of these cells than cell death. In addition, sensitivity analysis also demonstrated that the CCR5 gene editing CD45RA int RO int T SCM The growth rate of T EM The results also showed that the CCR5 gene-edited CD45RA cells had a significant positive correlation with the cell number of all CCR5 gene-edited cells, including the CD45RA cells, which was not observed for the proliferation rate of other subsets (Figure 6B). int RO int T SCM These findings suggest that self-renewal of CCR5 is important for the recruitment and maintenance of CCR5 mutations in other memory subsets.

[0226] In support of these findings, higher CCR5 gene-edited CD45RA before ART discontinuation (week 6) + RO - T SCM , CD45RA int RO int TSCM and T CM The cell counts were higher in CCR5 gene-edited T cells after ATI (week 22). EM Correlated with cell count, viremia was T SCM T EM This confirms that gradual differentiation into IFN-γ-γ cells can be induced (Figure 6C-E).

[0227] T EM T cells have been shown to express the highest levels of CCR5 compared to other memory cells, and therefore may be involved in viral replication. EM Maintaining a subset of CCR5 gene-edited cells within the 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 weeks 6 and 22 post-infusion. Our results suggest that T cells carrying integrated HIV DNA are more likely to be infected than CCR5 gene-edited. EM The frequency of T did not change significantly during ATI (Figure 6F). Closer examination revealed that 50% of the analyzed participants had T between weeks 6 and 22. EM The researchers found that increasing the size of the reservoir in cells resulted in the remaining half showing no change or decrease in the frequency of integrated HIV DNA. EM Changes in the frequency of cells harboring integrated HIV DNA within T EM This was inversely correlated with the frequency of CCR5 gene-edited alleles in the population (Figure 6G). EM We further found that a higher frequency of the CCR5 gene-edited allele at week 22 in a subset (but not in other subsets) specifically correlated with a greater reduction in viral load at week 22 compared to the historical pre-ART viral load set point (Figure 6H-I). Collectively, these results support the conclusion that CCR5 gene-edited T EM The continuous recruitment of T cells during viremia as a result of differentiation from their precursor cells EM Limiting the size of the reservoir in the EM The superior protection from de novo infection of a subset indicates an impact on the control of active viral replication during ATI.

[0228] Observations from this study suggest that the novel CD45RA int RO int T SCM This further supports our findings generated from the SB-728-0902 cohort, demonstrating that a subset of short-lived T cells expressing CCR5 mutations have the highest levels of CCR5 gene-edited alleles and can differentiate into other memory subsets. EM Differentiated memory T cells, including those from the HIV-1 family, are protected from viral infection, leading to the control of viremia and progressive attenuation of the HIV reservoir, as observed in both studies.

[0229] CD45RA+ T SCM The subset was previously described with 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 Although a CD45RA+CD45RO-like phenotype has previously been reported following in vitro expansion of purified CD4+ and CD8+ naive T cells costimulated in the presence of cytokines such as IL-2, IL-7, IL-15, or IL-21, the ability of these cells to persist in vivo and whether a proportion of these cells can revert to a CD45RA+CD45RO- phenotype have not been investigated. In addition, a CD4+ subset expressing low levels of CD45RA and CD45RO appears to emerge in vivo upon initiation of ART combined with IL-2 therapy, which correlates with CD4+ T cell expansion, and CD45RA int RO int This demonstrates that CD45RA cells can also be generated in vivo in response to homeostatic proliferation. int RO int T SCM The subset also demonstrated the capacity for self-renewal as observed by gene set enrichment of the Wnt signaling cascade, upregulation of additional mechanisms of stemness (SOX genes), and CD45RA+ T SCM Unlike the CD45RA subset, int ROint T SCM We further observed subset-specific enhanced pro-inflammatory and apoptotic signatures and identified these T SCM It further emphasized the distinction between groups.

[0230] T SCM have previously been shown to be permissive to HIV infection. The importance of limiting HIV infection with early memory cells to maintain CD4+ T cell homeostasis has been demonstrated not only in non-human primates but also in individuals with virally non-progressive HIV infection. T cells in secondary lymphoid tissues (enriched in CCR5 gene-edited alleles (up to 40% in the periphery at 3-4 years)) are SCM The presence of these cells (which partially, if not completely, inhibit HIV replication) may lead to their long-term survival. 。 This in turn leads to an overall improvement in adaptive immune function, an increase in CD4+ T cell counts, and control of HIV and other pathogens, resulting in a reduction in reservoir size, as shown in the current study. Indeed, we found that T TM and T EM T in SB-728-T product in short-lived cells such as SCM Detect subset-specific CCR5 gene-edited cells and SCM T EM Furthermore, these observations support modeling the homeostasis of CCR5 gene-edited CD4+ T cell memory subsets, with the observed reduction in mortality. Additionally, biphasic decay analysis of HIV DNA ruled out the possibility that dilution was the cause of HIV decay. However, our multivariate model did not support the CCR5 gene-edited CD45RA int RO int T SCM demonstrated that the long-term persistence of HIV 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 provides a global benefit, allowing control of viral replication. In support of this, the results of both the SB-728-0902 and SB-728-1101 studies confirmed the role of SB-728-T infusion in restoring T cell homeostasis and providing cognate help to HIV-specific CD8 T cells, resulting in a significant increase in T SCM of T, which protects against HIV infection EM The cognate help provided is further emphasized by the observed decay in the HIV reservoir, which expresses CD45RA int RO int T SCM Cell- and GAG-specific CD8+ T TM The role of IL-2 production by HIV-specific CD8+ T cells in viral replication has been previously demonstrated.

[0232] Our results demonstrate that CCR5 gene-edited and gene-unmodified T cells in the injected product SCM We recognized that this could be due to the ex vivo expansion of both CCR5-modified and unmodified cells, which are protected in vivo by ART. Supporting this hypothesis, we observed a clear expansion of both CCR5-modified and unmodified cells after infusion. Interestingly, our model demonstrated that CD45RA int RO int T SCM We show that the proliferation rate of CCR5 gene-edited cells was positively correlated with cell counts in all CCR5 gene-edited cells. However, it should be noted that previous studies using adoptively transferred anti-CD3 / CD28 costimulated CCR5-unmodified cells failed to sustainably increase CD4+ T cell counts in HIV+ participants. We are currently conducting a randomized clinical trial injecting CCR5-modified versus unmodified cells to more clearly address this issue.

[0233] The results described here demonstrate that a single infusion of CCR5 gene-edited cells is safe, well-tolerated, and can lead to a significant reduction in HIV DNA levels (and presumably the replication-competent HIV reservoir). Additionally, the long-term persistence of gene-edited memory stem cells allows for the replacement of old, dysfunctional / infected memory cells with new cells protected from infection, thereby repopulating the immune system. The non-invasive and autologous aspect of this therapy makes it more accessible and less cumbersome than hematopoietic stem cell transplantation. Advances in zinc finger nuclease mRNA delivery by electroporation enable multi-dose regimens, which are expected to significantly improve CD4+ T-cell counts. In line with this, multiple infusions of unmodified anti-CD3 / CD28 costimulated unmodified CD4+ T cells every 8 weeks previously demonstrated significant increases in cell counts 1 year after infusion. Additionally, as CD4+ T cell counts increase after each infusion, products from HIV+ subjects with high CD4+ T cell counts have been shown to expand better in vitro compared to subjects with low CD4+ T cell counts, and therefore subsequent products are expected to also result in better engraftment and persistence. Furthermore, statistical analysis of HIV DNA showed that 6 weeks of infusion partially reduced the reservoir, suggesting that optimal viral load control may be achieved if treatment is interrupted after an extended period along the second phase of decay.

[0234] In summary, our results demonstrate that infusion of CCR5 gene-edited cells offers a unique therapeutic intervention that improves T cell homeostasis and reduces the 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 preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details can be made therein 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 hereby incorporated by reference in their entirety.

Claims

1. 1. A method for producing an enriched population of long-lived CD4 T cells ex vivo that lack a functional CCR5 HIV coreceptor, said method comprising: isolating CD4 T cells from a biological sample of a subject; Isolating isolated CD4 T cells having a CD45RA int CD45RO int phenotype by sorting and / or ｉｎｔ CD45RO ｉｎｔ enriching in culture isolated CD4 T cells having a phenotype, wherein at least 50% of said separated and / or enriched CD4 T cell population has a CD45RA int CD45RO int CD95+CD127+CD27+ phenotype, and enriching in culture comprises culturing said isolated CD4 T cells in the presence of IL-7 and IL-15 to promote expansion and / or formation of an enriched population of CD4 T cells having a CD45RA int CD45RO int phenotype, wherein the concentration of IL-7 and IL-15 is less than 10 ng / ml; CD45RA ｉｎｔ CD45RO ｉｎｔ modifying the isolated CD4 T cells so that they lack a functional CCR5 HIV coreceptor and / or modifying the CD4 T cells so that they lack a functional CCR5 HIV coreceptor prior to sorting and / or enriching in culture to produce cells having a phenotype ｉｎｔ CD45RO ｉｎｔ The CD45RA phenotype is expressed in a manner that allows CD4 T cells to express the HIV-1 receptor phenotype without a functional CCR5 HIV coreceptor. ｉｎｔ CD45RO ｉｎｔ and modifying the isolated and / or enriched CD4 T cells with the phenotype.

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

3. The isolated and / or enriched CD4 T cells are CD45RA ｉｎｔ CD45RO ｉｎｔ The method of claim 1, wherein the cells have a CD95+CD127+CD27+TCF7+ phenotype.

4. The isolated and / or enriched CD4 T cells are CD45RA ｉｎｔ CD45RO ｉｎｔ 2. The method of claim 1, wherein the cells have a CD95+CD127+CD27+TCF7+IL7R+CD44+SCL38A1+IL2RG+CD6+CD5+ phenotype.

5. 2. The method of claim 1, further comprising activating the isolated CD4 T cells by culturing the isolated CD4 T cells with an anti-CD3 antibody and / or an anti-CD28 antibody.

6. CD45RA ｉｎｔ CD45RO ｉｎｔ To maintain the phenotype, the CD45RA ｉｎｔ CD45RO ｉｎｔ 10. The method of claim 1, further comprising culturing the isolated and / or enriched CD4 T cells having a phenotype.

7. 2. The method of claim 1, wherein the CD4 T cells lacking the functional CCR5 HIV coreceptor are modified by inactivating the gene encoding CCR5.

8. 8. The method of claim 7, wherein the isolated CD4 T cells are genetically modified by at least one of transduction, transfection, and / or electroporation.

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