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  • C12N2510/00—Genetically modified cells

Definitions

• enhancing the recovery of CD4+ T cells may contribute to the reduction of the HIV reservoir during ART.
• CCR5 is one of the major co-receptors for HIV entry.
• the therapeutic concept of providing HIV-infected subjects with a CCR5 deficient immune compartment was demonstrated with the“Berlin Patient”, who has been HIV-free since receiving allogeneic bone marrow transplants of CD34 + stem cells from a homozygous CCR5A32 matched donor. While these results are encouraging, a less invasive and a more broadly applicable treatment strategy would be desirable.
• One approach is to reconstitute immune function through adoptive transfer of autologous T cells, which was successfully deployed in other viral infections, including cytomegalovirus and Epstein-Barr virus, but largely failed in HIV infection, partly because CD4+ T cells remain susceptible to HIV infection.
• Embodiments described herein relate to a long-lived enriched population of CD4 T cells and CD 8 T cells (CD4/CD8 T cells) having a CD45RA int CD45RO int phenotype, genetically modified and/or altered CD4/CD8 T cells having a CD45RA int CD45RO int phenotype, and to their use in treating an HIV infected subject and particularly a latent HIV infection of a subject that has undergone and/or continues to undergo antiretroviral therapy.
• CD4/CD8 T cells has phenotypic and molecular attributes of long- lived pluripotent stem cells. Like other known stem cell populations, this subset population has a low metabolic profile (upregulation of fatty acid metabolism and oxidative
• CD45RA int CD45RO int CD45RA int CD45RO int
• CD4/CD8 T cells having a CD45RA int CD45RO int phenotype can also express CD95 (Fas) CD 127 (IL7R) and CD27. Addition of low doses of cytokines IL-7 and IL-15 can lead to the formation of an enriched population of CD4/CD8 cells having the CD45RA mt CD45RO mt phenotype; while high doses of cytokines IL-7 and IL-15 can lead to effector differentiation of the cells.
• CD4/CD8 T cells having a CD45RA mt CD45RO mt phenotype can be genetically modified such that they are devoid of a functional CCR5 and/or CXCR4 HIV co-receptor.
• Administration of CCR5 and/or CXCR4 gene edited autologous CD4/CD8 T cells having a CD45RA mt CD45RO mt phenotype to an HIV infected subject can provide sustained increases in CD4+ T cell counts, restored T cell homeostasis, and a sizable decline in the size of the HIV reservoir in the subject.
• a method of generating an enriched population of CD4/CD8 T cells having a CD45RA int CD45RO int phenotype, which can be genetically modified such that the CD4/CD8 T cells are devoid of a functional CCR5 and/or CXCR4 HIV co-receptor includes isolating T-cells from a biological sample of a subject.
• the biological sample can include a T cell containing sample, such as peripheral blood mononuclear cells, of a subject having 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
• a population of CD4/CD8 T cells having a CD45RA int CD45RO int phenotype can be separated from the isolated T-cells.
• the CD4/CD8 T cells can be genetically modified such that the CD4/CD8 T cells are devoid of a functional CCR5 and/or CXCR4 HIV co-receptor before separating the population of CD4/CD8 T cells having the CD45RA int CD45RO int phenotype from the isolated T-cells.
• the population of CD4/CD8 T cells having the CD45RA int CD45RO int phenotype can be genetically modified after separation from the isolated T-cells such that the population of CD4/CD8 T cells having the CD45RA int CD45RO int phenotype are devoid of a functional CCR5 and/or CXCR4 HIV co-receptor.
• the isolated CD4/CD8 T-cell are genetically modified by at least one of transduction, transfection, and/or electroporation to inactivate a gene encoding CCR5 and/or CXCR4 in the cells.
• the separated CD4/CD8 T cells can express at least one of CD95, CD127, or CD27. In other embodiments, the separated CD4/CD8 T cells can intermediately express 4- IBB and optionally express 0X40.
• the separated CD4/CD8 T-cells can express at least one of, at least two of, at least three of, at least four of, at least five of or more of IL17RA, CD5, IL2RG, IGF2R, SLC38A1, IL7R, SLC44A2, SLC2A3, CD96, CD44, CD6, CCR4, IL4R, or SLC12A7.
• the separated CD4/CD8 T cells can have a
• the separated T-cells can have a CD45RA int CD45RO int CD95+ CD127+CD27+IL7R+CD44+ SCL38A1+IL2RG+CD6+CD5+ phenotype.
• the method can include activating the isolated CD4/CD8 T cells with an anti-CD3 antibody and/or an anti-CD28 antibody prior to genetic modification and/or separation.
• the activated CD4/CD8 T cells can be cultured in an amount of IL7 and IL15 effective to promote expansion and/or formation of an enriched population of CD4/CD8 T cells having a CD45RA int CD45RO int phenotype.
• the CD4/CD8 T-cells can be cultured in a culture medium comprising TGFp/ILl b to maintain the
• CD45RA int CD45RO int phenotype CD45RA int CD45RO int phenotype
• compositions that includes an enriched population of CCR5 and/or CXCR4 gene edited CD4/CD8 T cells produced by a method described herein. At least about 70%, at least about 75%, at least about 80%, at least 85%, at least about 90%, at least about 95% of the enriched population of CCR5 and/or CXCR4 gene edited CD4/CD8 T cells can have a CD45RA mt CD45RO mt phenotype.
• the composition or enriched T-cell population can be administered to a subject with an HIV infection to treat the HIV infection.
• administration of the composition or enriched T-cell population to a subject with HIV is capable of promoting at least one of a sustained increase in absolute CD4 cell numbers, restoration of HIV specific T cell immunity, and a substantial decay in HIV reservoir in the subject.
• the subject has undergone and/or continues to undergo antiretroviral therapy.
• FIG. 1 is a flow diagram illustrating a method of generating an enriched population of CD4/CD8 T cells having a CD45RA int CD45RO int phenotype.
• Figs. 2(A-F) illustrate plots showing decay of the HIV reservoir post SB-728-T infusion correlates with persistence of CCR5 gene edited cells.
• A Box-plot with overlaid jitter showing the frequency of cells harboring total HIV DNA per 10 6 PBMCs at baseline (BL), year 1, and year 2 post infusion. Box shows median, first and third quartiles, and whiskers extend to maximum and minimum values. Individual data points are shown for all 9 participants with colors corresponding to the different cohorts (cohort 1, 2 and 3 are shown in blue, green and red hues, respectively).
• BL values for subjects 1-01 and 1-02 were imputed as described in the materials and methods. * P ⁇ 0.05; Wilcoxon rank-sum test.
• B Frequencies of integrated HIV DNA copies per 10 6 purified CD4+ T cells are shown at BL and year 2-3 (long-term follow up). Participants in cohorts 1, 2 and 3 are shown in blue, green and red symbols, respectively. * P ⁇ 0.05; Wilcoxon rank-sum test.
• C-D Association between the change in the frequency of PBMCs harboring total HIV DNA at long term time points (Ratio of log 10 values at day 720 over day 0) and the fold-expansion of Pentamer Duplication marked cells at day 21 (C) and years 3-4 post infusion (D). Scatterplots and predictions from robust regression models are shown along with the 95% confidence intervals (shaded regions).
• E Representative example of the bi-phasic decay analysis of the HIV DNA (participant 3-01) using Monolix, a software for parameter estimation in non linear mixed effect models.
• the blue line represents the bi-phasic exponential fitted line to the HIV DNA copy per 10 6 PBMC (represented by the red stars).
• the lines represent the fast and slow decay and the plateau reached after the end of the slow decay phase, respectively. Inserts highlight the two intersection points that represent the beginning and end of the slow phase decay.
• F Representative example of the estimated total HIV DNA per 10 6 PBMCs that are expected as a result of dilution (participant 3-01) post infusion (red line).
• Figs. 3(A-D) illustrate graphs and plots showing identification of a novel memory stem cell CD4+ T cell subset (CD45RA int RO int cells expressing CD95) that contributes to the persistence of CCR5 gene edited T cells and total CD4+ T cells but contributes minimally to the CD4+ T cell reservoir.
• N/A not done; limitations in cryopreserved PBMCs prevented quantification of TSCM subsets at early time points.
• Each dot in the scatter plot corresponds to a participant with dot size proportional to the HIV DNA day 720/BL ratio, with a greater decay symbolized by a smaller dot size.
• FIGs. 4(A-G) illustrate tables, graphs, and plots showing CD45RA mt RO mt TSCM are distinct from previously identified CD45RA + TSCM cells.
• a color gradient depicts the GSEA normalized enrichment score (NES ranging from -4 to +5) of pathways enriched in genes induced or repressed in CD45RA mt RO mt TSCM compared to TEM and TCM (P ⁇ 0.05).
• GSEA Gene Set Enrichment Analysis
• Figs. 5(A-H) illustrate plots showing CCR5 gene edited TSCM prior to ATI correlate with control of viral load.
• A Plot depicting the viral load (VL) values at week 22 (equivalent to 16 weeks of ATI) and the historic pre-ART viral set point values obtained from participants’ charts (data available for 14 out of 15 participants from study 1101 cohorts 1-5). Participants with extended ATI are shown in red. P value of Wilcoxon rank-sum test is shown.
• B-C Spearman rank correlations between the change of VL between week 22 and historic pre-ART viral set point with the change in CD4+ T cell counts during peak expansion (weeks 1-3 post infusion) (B) and with the frequency of the“Pentamer
• Figs. 6(A-I) illustrate plots showing levels of CCR5 gene edited TEM during ATI correlate with control of viral load and lower reseeding of the TEM HIV reservoir.
• A Box-plot showing the percent of ZFN-induced CCR5 mutations present uniquely in
• B Schematic figure showing the dynamics of the CCR5 gene edited CD4+ T-cell dynamics (see Material & Methods for full model details and assumptions). Model
• Parameters are obtained by taking the geometrical mean of the 5 individual fitting results of the five participants with an extended ATI period. Parameters listed in the boxes indicate the parameters that expressed a significant correlation (> ⁇ 0.5) with the cell population magnitude obtained from the sensitivity analysis test performed in MATLAB using 100,000 bins.
• Fig. 7 illustrates a plot showing the size of the HIV reservoir in SB-728-0902 study participants at baseline. Correlation between CD4+ T cell counts at baseline (BL) and levels of integrated HIV DNA at BL measured in purified CD4+ T cells. Spearman’s rho (p) test was used. Dashed lines represent the 95% confidence bands.
• Figs. 8(A-E) illustrate a single SB-728-T infusion led to a sustained increase in total CD4+ T cell counts, amelioration of the CD4:CD8 ratio, and long-term persistence of CCR5 gene edited cells.
• A, CD4+ T cell counts are shown at baseline (BL; 7 days prior to infusion), day 14, months 3, 6, and 12, as well as for long-term follow up time points including year 2 and a last follow up at years 3-4. The mean is shown in a black line.
• Duplication-marked CD4+ T cells following infusion was estimated, as described in Material & Methods, for all 9 study participants during follow up.
• the grey area represents data points with a fold change below 1.
• D Box-plot with overlaid jitter of Pentamer Duplication (marker of gene edited cells) per 10 6 mononuclear cells from rectal biopsies post infusion. Box-plots show the 75th (upper edge), median (solid line in the box), and 25th percentile (lower edge). Whiskers are drawn from minimum to maximum values.
• Figs. 9(A-C) illustrate plots showing characterization of SB-728-T products.
• A Levels of integrated HIV DNA in purified CD4+ T cells from pre-manufacture leukapheresis samples (BL) and post manufacture (SB-728-T products). Wilcoxon signed rank test P values shown.
• Live CD3+ CD4+ cells were gated on CD45RA and CD45RO, followed by CCR7 and CD27 to identify naive (CD45RA+CD45RO-CCR7+CD27+), CD45RA int CD45RO int Tsc M -like cells, TCM (CD45RA-CD45RO+CCR7+CD27+), T TM (CD45RA-CD45RO+CCR7- CD27+), TEM (CD45RA-CD45RO+CCR7-CD27-), and CD45RA-CD45RO+CCR7+CD27- subsets.
• B Frequencies of CD4+ T cell subsets observed in SB-728-T products. Lines represent the mean and standard deviation.
• Figs. 10(A-F) illustrate plots showing frequencies of CD58+CD95+ cells in CD45RA mt CD45RO mt and CD45RA + CD45RO subsets increase post infusion and contribute to persistence of CCR5 gene edited cells.
• A-B Histograms showing the mean frequencies of cells expressing CD58 and CD95 in the CD45RA mt CD45RO mt
• CD45RA int RO int CD95 cell counts at BL (up to 3 months before infusion), and at mid (month 6), late (months 8-11), and long-term time points (up to month 44) post-infusion for the 6 subjects in which BL analysis was performed.
• Fig. 1 1 illustrates a plot showing CD45RA mt RO mt TSCM cells have higher levels of CCR5 gene edited alleles compared to other memory subsets. Box-plot with overlaid jitter of the frequencies of CCR5 gene edited alleles within sorted CD4+ T cell subsets at year 2-4 post infusion, measured by DNA sequencing of the diverse CCR5 ZFN-induced mutations. Lines represent the mean and standard deviation. Box plots show the 75th (upper edge), median (solid line in the box), and 25th percentile (lower edge). Whiskers are drawn from minimum to maximum values. Wilcoxon signed rank test P values shown.
• Figs. 12(A-B) illustrate plots showing CCR5 gene edited T cells contribute to a polyclonal unbiased reconstitution of T cells.
• Fig. 14(A-B) illustrate plots showing CD45RA int RO int TSCM cells constitute a distinct population than the previously described CD45RA + TSCM subset.
• Fig. 15(A-B) illustrate plots showing viral loads of subjects who underwent analytical treatment interruption (ATI) post SB-728-T infusion in the 1101 study.
• A
• Figs. 16(A-B) illustrate plots showing CD4+ T cell subset distribution and cell counts post infusion (week 6) and post treatment interruption (week 22) in the 1101 study.
• Fig. 17 illustrates Frequencies of CCR5 gene edited cells in CD4+ T cell subsets post ATI in the 1101 study.
• the frequency of CCR5 gene edited alleles, determined by DNA sequencing, is shown for CD4+ T cell subsets (CD45RA+ TSCM, CD45RA int RO int TSCM, TCM, and TEM) at week 6 (pre-ATI), week 22, month 7/8, and month 12 post infusion (during ATI) for participants who had extended ATI until at least month 12.
• Activation refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions.
• activated T cells refers to, among other things, T cells that are undergoing cell division.
• antibody refers to an immunoglobulin molecule, which is able to specifically bind to a specific epitope on an antigen.
• Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoactive portions of intact immunoglobulins.
• Antibodies are typically tetramers of immunoglobulin molecules.
• the antibodies in the present invention may exist in a variety of 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).
• antigen or "Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
• antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein.
• an antigen need not be encoded solely by a 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 more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
• autologous is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.
• Allogeneic refers to a graft derived from a different animal of the same species.
• Xenogeneic refers to a graft derived from an animal of a different species.
• an "effective amount” as used herein means an amount which provides a therapeutic or prophylactic benefit.
• expression is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
• the term "specifically binds,” as used herein, is meant a molecule, such as an antibody, which recognizes and binds to another molecule or feature, but does not substantially recognize or bind other molecules or features in a sample.
• inhibitor means to reduce a molecule, a reaction, an interaction, a gene, an mRNA, and/or a protein's expression, stability, function or activity by a measurable amount or to prevent entirely.
• Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein, a gene, and an mRNA stability, expression, function and activity, e.g., antagonists.
• nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form.
• polynucleotide refers to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form.
• these terms are not to be construed as limiting with respect to the length of a polymer.
• the terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties
• an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T.
• polypeptide peptide
• protein protein
• amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally-occurring amino acids.
• 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).
• binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence- specific.
• Such interactions are generally characterized by a dissociation constant (K d ) of 10 6 M 1 or lower.
• K d dissociation constant
• Affinity refers to the strength of binding: increased binding affinity being correlated with a lower K d .
• chimeric RNA refers to the polynucleotide sequence comprising the guide sequence, the tracr sequence and the tracr mate sequence.
• guide sequence refers to the about 10-30 (10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
• Complementarity refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non- traditional types.
• a percent complementarity indicates the percentage of residues in a nucleic acid molecule which 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 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
• Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
• 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.
• a "binding protein” is a protein that is able to bind to another molecule.
• a binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein) and/or a protein molecule (a protein-binding protein).
• a DNA-binding protein a DNA-binding protein
• an RNA-binding protein an RNA-binding protein
• a protein-binding protein In the case of a protein-binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more molecules of a different protein or proteins.
• a binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding and protein-binding activity.
• a "zinc finger DNA binding protein” (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion.
• the term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.
• a "TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains/units. The repeat domains are involved in 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 with other TALE repeat sequences within a naturally occurring TALE protein.
• Zinc finger and TALE binding domains can be "engineered” to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger or TALE protein. Therefore, engineered DNA binding proteins (zinc fingers or TALEs) are proteins that are non-naturally occurring. Non-limiting examples of methods for engineering DNA-binding proteins are design and selection. A designed DNA binding protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP and/or TALE designs and binding data. See, for example, U.S. Pat. Nos.
• a "selected" zinc finger protein or TALE is a protein not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. See e.g., U.S. Pat. Nos. 8,586,526; 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,200,759; as well as WO 95/19431; WO 96/06166; WO 98/53057;
• Recombination refers to a process of exchange of genetic information between two polynucleotides.
• HR homologous recombination
• This process requires nucleotide sequence homology, uses a "donor” molecule to template repair of a "target” molecule ( i.e the one that experienced the double-strand break), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target.
• such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or "synthesis- dependent strand annealing," in which the donor is used to re-synthesize genetic information that will become part of the target, and/or related processes.
• Such specialized HR often results in an 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.
• one or more targeted nucleases are provided.
• a double- stranded break in the target sequence e.g., cellular chromatin
• a "donor" polynucleotide having homology to the nucleotide sequence in the region of the break, can be introduced into the cell.
• the presence of the double-stranded break has been shown to facilitate integration of the donor sequence.
• the donor sequence may be physically integrated or, alternatively, the donor polynucleotide is used as a template for repair of the break via homologous recombination, resulting in the introduction of all or part of the nucleotide sequence as in the donor into the cellular chromatin.
• a first sequence in cellular chromatin can be altered and, in certain embodiments, can be converted into a sequence present in a donor
• replace or replacement can be understood to represent replacement of one nucleotide sequence by another, ( i.e ., replacement of a sequence in the informational sense), and does not necessarily require physical or chemical
• additional CRISPR/Cas nucleases and/or additional pairs of zinc-finger or TALEN proteins can be used for additional double- stranded cleavage of additional target sites within the cell.
• a chromosomal sequence is altered by homologous recombination with an exogenous "donor" nucleotide sequence.
• homologous recombination is stimulated by the presence of a double-stranded break in cellular chromatin, if sequences homologous to the region of the break are present.
• the exogenous nucleotide sequence can contain sequences that are homologous, but not identical, to genomic sequences in the region of interest, thereby stimulating homologous recombination to insert a non-identical sequence in the region of interest.
• portions of the donor sequence that are homologous to sequences in the region of interest exhibit between about 80 to 99% (or any integer therebetween) sequence identity to the genomic sequence that is replaced.
• the homology between the donor and genomic sequence is higher than 99%, for example if only 1 nucleotide differs as between donor and genomic sequences of over 100 contiguous base pairs.
• a non-homologous portion of the donor sequence can contain sequences not present in the region of interest, such that new sequences are introduced into the region of interest.
• the non-homologous sequence is generally flanked by sequences of 50-1,000 base pairs (or any integral value therebetween) or any number of base pairs greater than 1,000, that are homologous or identical to sequences in the region of interest.
• the donor sequence is non-homologous to the first sequence, and is inserted into the genome by non-homologous recombination mechanisms.
• Any of the methods described herein can be used for partial or complete inactivation of one or more target sequences in a cell by targeted integration of donor sequence that disrupts expression of the gene(s) of interest.
• Cell lines with partially or completely inactivated genes are also provided.
• the methods of targeted integration as described herein can also be used to integrate one or more exogenous sequences.
• the exogenous nucleic acid sequence can comprise, 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).
• the exogenous nucleic acid sequence may produce one or more RNA molecules (e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs (miRNAs), etc.).
• Cleavage refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double- stranded cleavage are possible, and double- stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, fusion polypeptides are used for targeted double- stranded DNA cleavage.
• a "cleavage half-domain” is a polypeptide sequence which, in conjunction with a second polypeptide (either identical or different) forms a complex having cleavage activity (preferably double-strand cleavage activity).
• 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 cleavage half-domains that dimerize.
• An "engineered cleavage half-domain” is a cleavage half-domain that has been modified so as to form obligate heterodimers with another cleavage half-domain
• sequence refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double stranded.
• donor sequence refers to a nucleotide sequence that is inserted into a genome.
• a donor sequence can be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value therebetween or thereabove), preferably between about 100 and 1,000 nucleotides in length (or any integer therebetween), more preferably between about 200 and 500 nucleotides in length.
• a "homologous, non-identical sequence” refers to a first sequence which shares a degree of sequence identity with a second sequence, but whose sequence is not identical to that of the second sequence.
• a polynucleotide comprising the wild-type sequence of a mutant gene is homologous and non-identical to the sequence of the mutant gene.
• the degree of homology between the two sequences is sufficient to allow homologous recombination therebetween, utilizing normal cellular mechanisms.
• Two homologous non-identical sequences can be 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 or more kilobases
• Two polynucleotides comprising the homologous non-identical sequences need not be the same length.
• an exogenous polynucleotide i.e., donor polynucleotide
• an exogenous polynucleotide i.e., donor polynucleotide
• 20 and 10,000 nucleotides or nucleotide pairs can be used.
• nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this fashion. In general, identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides 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 sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.
• An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986).
• An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility
• Suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.
• sequences described herein the range of desired degrees of sequence identity is approximately 80% to 100% and any integer value therebetween.
• percent identities between sequences are at least 70-75%, preferably 80-82%, more preferably 85-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity.
• the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that allow formation of stable duplexes between homologous regions, followed by digestion with single- stranded- specific nuclease(s), and size determination of the digested fragments.
• Two nucleic acid, or two polypeptide sequences are substantially homologous to each other when the sequences exhibit at least about 70%-75%, preferably 80%-82%, more preferably 85%-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity over a defined length of the molecules, as determined using the methods above.
• substantially homologous also refers to sequences showing complete identity to a specified DNA or polypeptide sequence.
• DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is known to those with skill of the art. See, e.g., Sambrook et ah, supra; Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington,
• 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 hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit the hybridization of a completely identical sequence to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art
• Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe 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.
• a partial degree of sequence identity for example, a probe having less than about 30% sequence identity with the target molecule
• Chromatin is the nucleoprotein structure comprising the cellular genome.
• Cellular chromatin comprises nucleic acid, primarily DNA, and protein, including histones and non-histone chromosomal proteins.
• the majority of eukaryotic cellular chromatin exists in the form of nucleosomes, wherein a nucleosome core comprises approximately 150 base pairs of DNA associated with an octamer comprising two each of histones H2A, H2B, H3 and H4; and linker DNA (of variable length depending on the organism) extends between nucleosome cores.
• a molecule of histone HI is generally associated with the linker DNA.
• chromatin is meant to encompass all types of cellular nucleoprotein, both prokaryotic and eukaryotic.
• Cellular chromatin includes both chromosomal and episomal chromatin.
• a "chromosome,” is a chromatin complex comprising all or a portion of the genome of a cell.
• the genome of a cell is often characterized by its karyotype, which is the collection of all the chromosomes that comprise the genome of the cell.
• the genome of a cell can comprise one or more chromosomes.
• An "episome” is a replicating nucleic acid, nucleoprotein complex or other structure comprising a nucleic acid that is not part of the chromosomal karyotype of a cell.
• Examples of episomes include plasmids and certain viral genomes.
• An "accessible region” is a site in cellular chromatin in which a target site present in the nucleic acid can be bound by an exogenous molecule which recognizes the target site. Without wishing to be bound by any particular theory, it is believed that an accessible region is one that is not packaged into a nucleosomal structure. The distinct structure of an accessible region can often be detected by its sensitivity to chemical and enzymatic probes, for example, nucleases.
• 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.
• An "exogenous" molecule is a molecule that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods.
• Normal presence in the cell is determined with respect to the particular developmental stage and environmental conditions of the cell.
• a molecule that is present only during embryonic development of muscle is an exogenous molecule with respect to an adult muscle cell.
• a molecule induced by heat shock is an exogenous molecule with respect to a non-heat-shocked cell.
• An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally-functioning endogenous molecule.
• An exogenous molecule can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules.
• Nucleic acids include DNA and RNA, can be single- or double-stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Pat. 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.
• transgenes or "genes of interest” which are exogenous sequences introduced into a cell.
• an exogenous molecule can be the same type of molecule as an endogenous molecule, e.g., an exogenous protein or nucleic acid.
• an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell.
• Methods for the introduction of exogenous molecules into cells include, but are not limited to, lipid-mediated transfer (i.e ., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer.
• exogenous molecule can also be the same type of molecule as an endogenous molecule but derived from a different species than the cell is derived from.
• a human nucleic acid sequence may be introduced into a cell line originally derived from a mouse or hamster.
• Methods for the introduction of exogenous molecules into plant cells include, but are not limited to, protoplast transformation, silicon carbide (e.g., WHISKERS.
• TM. TM.
• Agrobacterium-mediated transformation lipid-mediated transfer (i.e., liposomes, including 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.
• an "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions.
• an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally-occurring episomal nucleic acid. Additional endogenous molecules can include proteins, for example, transcription factors and enzymes.
• the term "product of an exogenous nucleic acid" includes both polynucleotide and polypeptide products, for example, transcription products
• RNA Ribonucleotides
• translation products polypeptides
• a "fusion" molecule is a molecule in which two or more subunit molecules are linked, preferably covalently.
• the subunit molecules can be the same chemical type of molecule, or can be different chemical types of molecules.
• Examples of the first type of fusion molecule include, but are not limited to, fusion proteins (for example, a fusion between a ZFP or TALE DNA-binding domain and one or more activation domains) and fusion nucleic acids (for example, a nucleic acid encoding the fusion protein described supra).
• fusion polypeptide is a polypeptide comprising a polypeptide or portion ( e.g one or more domains) thereof fused or bonded to heterologous polypeptide.
• fusion polypeptides include immunoadhesins which combine a portion of the Cas protein with an immunoglobulin sequence, and epitope tagged
• polypeptides which may comprise a Cas protein, for example, or portion thereof fused to a "tag polypeptide".
• the tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with nuclease activity of Cas.
• Suitable tag polypeptides generally have at least six amino acid residues and usually between about 6-60 amino acid residues.
• Fusion protein in a cell can result from delivery of the fusion protein to the cell or by delivery of a polynucleotide encoding the fusion protein to a cell, wherein the polynucleotide is transcribed, and the transcript is translated, to generate the fusion protein.
• Trans- splicing, polypeptide cleavage and polypeptide ligation can also be involved in expression of a protein in a cell. Methods for polynucleotide and polypeptide delivery to cells are presented elsewhere in this disclosure.
• An “engineered gene” refers to a gene which has been altered in some manner such that it is non-identical with a wild type gene.
• Alterations can be in the form of targeted deletions, insertions and truncations.
• An engineered gene can comprise coding sequences from two heterologous genes or may comprise synthetic gene sequences.
• An engineered gene may also comprise changes in the coding sequence that are silent in the protein sequence (e.g ., codon
• An engineered gene can also comprise a gene with altered regulatory sequences.
• Gene expression refers to the conversion of the information, contained in a gene, into a gene product.
• a gene product can be the direct transcriptional 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 which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
• Modulation of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. Genome editing (e.g., cleavage, alteration, inactivation, random mutation) can be used to modulate expression. Gene inactivation refers to any reduction in gene expression as compared to a cell that does not include a CRISPR/Cas system as described herein. Thus, gene inactivation may be partial or complete.
• a "region of interest” is any region of cellular chromatin, such as, for example, a gene or a non-coding sequence within or adjacent to a gene, in which it is desirable to bind an exogenous molecule. Binding can be for the purposes of targeted DNA cleavage and/or targeted recombination.
• a region of interest can be present in a chromosome, an episome, an organellar genome (e.g., mitochondrial, chloroplast), or an infecting viral genome, for example.
• a region of interest can be within the coding region of a gene, within transcribed non-coding regions such as, for example, leader sequences, trailer sequences or introns, or within non-transcribed regions, either upstream or downstream of the coding region.
• a region of interest can be as small as a single nucleotide pair or up to 2,000 nucleotide pairs in length, or any integral value of nucleotide pairs.
• "Eukaryotic" cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells (e.g., T-cells).
• operative linkage and "operatively linked” (or “operably linked”) are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components.
• a transcriptional regulatory sequence such as a promoter
• a transcriptional regulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it.
• an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence, even though they are not contiguous.
• the term "operatively linked" can refer to the fact that each of the components performs the same function in linkage to the other component as it would if it were not so linked.
• the Cas DNA-binding domain and the activation domain are in operative linkage if, in the fusion polypeptide, the Cas DNA-binding domain portion is able to bind its target site and/or its binding site, while the activation domain is able to up-regulate gene expression.
• the Cas DNA-binding domain and the cleavage domain are in operative linkage if, in the fusion polypeptide, the Cas DNA-binding domain portion is able to bind its target site and/or its binding site, while the cleavage domain is able to cleave DNA in the vicinity of the target site.
• 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, yet retains the same function as the full-length protein, polypeptide or nucleic acid.
• a functional fragment can possess 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.
• DNA-binding function of a polypeptide can be determined, for example, by filter-binding, electrophoretic mobility- shift, or immunoprecipitation assays. DNA cleavage can be assayed 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 complementation, both genetic and biochemical. See, for example, Fields et al. (1989) Nature 340:245-246;
• a “vector” is capable of transferring gene sequences to target cells.
• vector construct means any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells.
• vector transfer vector means any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells.
• the term includes cloning, and expression vehicles, as well as integrating vectors.
• reporter gene refers to any sequence that produces a protein product that is easily measured, preferably although not necessarily in a 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, lucif erase), and proteins which mediate enhanced cell growth and/or gene
• 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 that encode reporters that may be operably linked to a desired gene sequence in order to monitor expression of the gene of interest.
• 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. Accordingly, the term “subject” or “patient” as used herein means any mammalian patient or subject to which the or stem cells of the invention can be administered. Subjects of the present invention include those that have been exposed to one or more chemical toxins, including, for example, a nerve toxin.
• the term "therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.
• the term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician.
• the term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, 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 to be treated.
• To "treat" a disease as the term is used herein means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.
• transfected or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
• a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
• the cell includes the primary subject cell and its progeny.
• a "vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
• vectors are known in the art including, but not limited to, linear polynucleotides,
• polynucleotides associated with ionic or amphiphilic compounds plasmids, and viruses.
• vector includes an autonomously replicating plasmid or a virus.
• the term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
• viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
• Ranges throughout this disclosure, various aspects of the invention can 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 the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
• Embodiments described herein relate to a long-lived enriched population of CD4 T cells and CD 8 T cells (CD4/CD8 T cells) having a CD45RA int CD45RO int phenotype, genetically modified and/or altered CD4/CD8 T cells having a CD45RA int CD45RO int phenotype, and to their use in treating an HIV infected subject and particularly a latent HIV infection of a subject that has undergone and/or continues to undergo antiretroviral therapy.
• CD4/CD8 T cells has phenotypic and molecular attributes of long- lived pluripotent stem cells. Like other known stem cell populations, this subset population has a low metabolic profile (upregulation of fatty acid metabolism and oxidative
• CD45RA int CD45RO int CD45RA int CD45RO int
• CD4/CD8 T cells having a CD45RA int CD45RO int phenotype can also express CD95 (Fas) CD 127 (IL7R) and CD27. Addition of low doses of cytokines IL-7 and IL-15 can lead to the formation of an enriched population of CD4/CD8 cells having the CD45RA mt CD45RO mt phenotype; while high doses of cytokines IL-7 and IL-15 can lead to effector differentiation of the cells.
• CD4/CD8 T cells having a CD45RA mt CD45RO mt phenotype can be genetically modified such that they are devoid of a functional CCR5 and/or CXCR4 HIV co-receptor.
• Administration of CCR5 and/or CXCR4 gene edited autologous CD4/CD8 T cells having a CD45RA int CD45RO int phenotype to an HIV infected subject can provide sustained increases in CD4+ T cell counts, restored T cell homeostasis, and a substantial decline in the size of the HIV reservoir in the subject.
• the enriched population CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having a CD45RA int CD45RO int phenotype upon transplantation or administration to a subject have the ability to persist or survive long term in the subject.
• the persistence can correlate with the efficacy of a therapeutic T cell transplant in the treatment of a disease, such as an HIV infection.
• long-lived, self-renewing and pluripotent CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having a CD45RA int CD45RO int phenotype can have a reduced cost of production, promote effector differentiation, and increase efficiency of treating latent HIV infection in a subject.
• frequencies of these cells in the currently available HIV therapy products can be used as a biomarker and be predictive of successful intervention.
• the enriched population CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having a CD45RA int CD45RO int phenotype 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 mt CD45RO mt phenotype after administration to a subject.
• the enriched population CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype can also possess an increased ability to engraft in a subject after administration.
• the enriched population CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype can possess an increased ability to engraft in a non-conditioned recipient.
• engraftment refers to the ability of the transplanted cells to populate a recipient and survive in the immediate aftermath of their transplantation. Accordingly, engraftment is assessed in the short term after transplantation. For example, engraftment may refer to the number of cells descended from the transplanted cells that are detected in the first in vivo evaluation of an experiment, clinical trial or therapeutic protocol, e.g., at the earliest time point that transplanted cells or their descendants may be detected in a recipient. In one embodiment, engraftment is assessed at 0-12, 0-24, 0-48 or 0-72 h after transplantation. In another embodiment, engraftment is assessed at about 1, 2, 3, 4, 5, 6, 12, 24, 36, 48, 60 or 72 h after transplantation. In a preferred embodiment, engraftment is assessed at about 12 h after transplantation.
• Fig. 1 illustrates a flow diagram illustrating a method of generating an enriched population of CD4/CD8 T cells having a CD45RA int CD45RO int phenotype, which can be genetically modified such that they are devoid of a functional CCR5 and/or CXCR4 HIV co receptor.
• 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 the subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
• the subject is a human.
• T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, and tumors.
• the T cells can be obtained from a subject having HIV to be treated, i.e., autologous T-cells from the subject to be treated.
• T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as ficoll separation.
• cells from the circulating blood of an individual are obtained by apheresis or leukapheresis.
• the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
• the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
• the cells can be washed with phosphate buffered saline (PBS).
• PBS phosphate buffered saline
• the wash solution lacks calcium and may lack magnesium or may lack many or all divalent cations.
• the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS.
• the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
• T cells can be isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL gradient.
• T cells can be isolated from umbilical cord.
• a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.
• the isolated T cells can include CD4+ T cells and/or CD8+ T cells.
• CD4 T cells and/or CD8 T cells can be isolated from the biological sample by positive or negative selection. Negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells.
• One method is cell sorting and/or selection via negative magnetic
• a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8.
• the concentration of cells and surface can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together ( i.e increase the concentration of cells), to ensure maximum contact of 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 cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
• a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.
• the isolated CD4/CD8 T cells can be activated and/or expanded by any suitable method known in the art.
• the T cells are activated and the numbers of T cells are 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-lb, 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-b, IFNa, IFNy, TNFa) recombinant proteins, costimulatory molecules, lectins, ionophores, synthetic molecules, antigen presenting cells (APCs), artificial APCs or feeders.
• APCs antigen presenting cells
• the CD4/CD8 T cells can activated and the numbers of T cells are 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 may be used in the inventive methods.
• non-specific T cell stimuli include anti-CD3 antibodies and anti- CD28 antibodies.
• the non-specific T cell stimulus may be anti-CD3 antibodies and anti-CD28 antibodies conjugated to beads.
• Any one or more cytokines may be used in the inventive methods.
• Exemplary cytokines include interleukin (IL)-2, IL-7, IL- 21, and IL-15.
• the CD4/CD8 T cells can be separated or sorted using, for example, flow cytometry, into an enriched population of CD4/CD8 T cells characterized by intermediate co-expression of CD45RA and CD45RO (CD45RA mt CD45RO mt ).
• the method may comprise sorting the cells in any suitable manner.
• the sorting is carried out using flow cytometry.
• the flow cytometry may be carried out using any suitable method known in the art.
• the flow cytometry may employ any suitable antibodies and stains.
• the flow cytometry is polychromatic flow cytometry.
• the enriched population of CD4/C8 T cells having a CD45RA int CD45RO int phenotype produced by the processes described herein can include CD4/C8 T cells having a CD45RA mt CD45RO mt as the majority cell type.
• the processes described herein produce cell cultures and/or cell populations comprising at 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%, 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
• the cells of the cell cultures or cell populations comprise human cells.
• the long lived CD4/CD8 T cells having a CD45RA int CD45RO int phenotype can also be characterized by the expression of other cell surface markers.
• the separated CD4/CD8 T cells having a CD45RA int CD45RO int phenotype can express at least one of CD95, CD127, or CD27.
• the CD4/CD8 T cells having a CD45RA mt CD45RO mt phenotype can further intermediately express 4- IBB and optionally express 0X40.
• the separated CD4/CD8 T-cells having a
• CD45RA mt CD45RO mt phenotype can further express at least one of, at least two of, at least three of, at least four of, at least five of or more of IL17RA, CD5, IL2RG, IGF2R, SLC38A1, IL7R, SLC44A2, SLC2A3, CD96, CD44, CD6, CCR4, IL4R, or SLC12A7.
• the separated CD4/CD8 T cells can have a
• the separated T-cells can have a CD45RA int CD45RO int CD95+
• the isolated CD4-CD8 T cells having the CD45RA mt CD45RO mt phenotype can be enriched by culturing the isolated CD4/CD8 T cells in a culture medium that includes low amount of IL-7 and/or IL-15.
• activated CD4/CD8 T cells cultured in low IL-7/IL-15 conditions can promote or form an enriched population of the CD4/C8 T cells having a CD45RA int CD45RO int phenotype compared to activated CD4/CD8 T cells cultured in high IL-7/IL-15 conditions (e.g., concentration of IL-7/IL-15 greater than 10 ng/ml).
• the culture medium can include IL-7 and/or IL-15 at a concentration, for example, of 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
• cell populations or cell cultures can be enriched in CD4/C8 T cells having a
• CD45RA mt CD45RO mt phenotype content by at least about 2- to about 1000-fold as compared to untreated cell populations or cell cultures.
• CD4/C8 T cells having a CD45RA mt CD45RO mt phenotype can be enriched by at least about 5- to about 500-fold as compared to untreated cell populations or cell cultures.
• CD4/C8 T cells having a CD45RA int CD45RO int phenotype can be enriched from at least about 10- to about 200-fold as compared to untreated cell populations or cell cultures.
• CD4/C8 T cells having a CD45RA int CD45RO int phenotype can be enriched from at least about 20- to about 100-fold as compared to untreated cell populations or cell cultures. In yet other embodiments, CD4/C8 T cells having a CD45RA int CD45RO int phenotype can be enriched from at least about 40- to about 80-fold as compared to untreated cell populations or cell cultures. In certain embodiments, CD4/C8 T cells having a
• CD45RA mt CD45RO mt phenotype can be enriched from at least about 2- to about 20-fold as compared to untreated cell populations or cell cultures.
• the CD4/CD8 T-cells can be cultured in a culture medium comprising TGFp/ILl b to maintain the CD45RA int CD45RO int phenotype.
• the addition of TGFP and/or IL1 b to the CD4/CD8 cells having a CD45RA mt CD45RO mt phenotype can lead to the maintenance of the CD45RA mt CD45RO mt phenotype prior to administration to a subject.
• the method can further include genetically modifying the CD4/CD8 T cells prior to or after activation and/or separation such that they are devoid of a functional CCR5 and/or CXCR4 HIV co-receptor.
• HIV infects human T cells, it relies on association with the T cell receptor CD4 and one of two co-receptors, the chemokine receptor CCR5 or CXCR4, to gain entry into the cell.
• Natural CCR5 variants (“CCR5-A32") in the human population were identified who appear to be resistant to HIV infection, especially in the homozygous state.
• disruption of one or both of the co-receptors may be accomplished to render the cell resistant to the vims (see U.S. Pat. No. 7,951,925).
• HIV patient T cells are edited at the CCR5 locus ex vivo to knock out the CCR5 gene. These cells are then re-introduced into the patient to treat HIV.
• the CD4/CD8 T cells can be genetically modified such that the CD4/CD8 T cells are devoid of a functional CCR5 and/or CXCR4 HIV co-receptor before separating the population of CD4/CD8 T cells having the CD45RA int CD45RO int phenotype from the isolated T-cells.
• the population of CD4/CD8 T cells having the CD45RA mt CD45RO mt phenotype can be genetically modified after separation from the isolated T-cells such that the population of CD4/CD8 T cells having the CD45RA mt CD45RO mt phenotype are devoid of a functional CCR5 and/or CXCR4 HIV co receptor.
• the genetic modification or genome editing of the CD4/CD8 T cells may be performed by transduction, transfection or electroporation.
• Transduction can performed with lentiviruses, gamma-, alpha-retroviruses or adenoviruses or with electroporation or transfection by nucleic acids (DNA, mRNA, miRNA, antagomirs, ODNs), proteins, site-specific nucleases (zinc finger nucleases, TALENs, CRISP/R), self replicating RNA viruses (e.g., equine encephalopathy vims) or integration-deficient lentiviral vectors.
• nucleic acids DNA, mRNA, miRNA, antagomirs, ODNs
• proteins protein
• site-specific nucleases zinc finger nucleases, TALENs, CRISP/R
• self replicating RNA viruses e.g., equine encephalopathy vims
• integration-deficient lentiviral vectors e.g., integration-deficient lentiviral vectors.
• the CD4/CD8 T cells and/or the CD4/CD8 T cells having the CD45RA int CD45RO int phenotype can be genetically modified by genome editing with engineered nucleases.
• Genome editing is a process of inserting, deleting, or modifying genomic sequences using sequence- specific nucleases.
• nucleases induce double- stranded DNA breaks that can subsequently be repaired by either non-homologous end joining (NHEJ) or homology dependent repair (HDR), which allows for insertion or modification of a gene using a template with homology to the DNA surrounding the double- stranded break.
• NHEJ non-homologous end joining
• HDR homology dependent repair
• genome editing has been performed by transfecting or transducing cells with RNA or DNA that then produce the proteins and, in the case of the CRISPR-Cas system, the guide RNAs, required for genome editing.
• a double-strand break (DSB) for can be created by a site-specific nuclease such as a zinc-finger nuclease (ZFN) or TAL effector domain nuclease (TALEN).
• ZFN zinc-finger nuclease
• TALEN TAL effector domain nuclease
• CRISPR/Cas systems are found in 40% of bacteria and 90% of archaea and differ in the complexities of their systems. See, e.g., U.S. Pat. No. 8,697,359.
• the CRISPR loci (clustered regularly interspaced short palindromic repeat) is a region within the organism's genome where short segments of foreign DNA are integrated between short repeat palindromic sequences. These loci are transcribed and the RNA transcripts (“pre-crRNA”) are processed into short CRISPR RNAs (crRNAs).
• CRISPR/Cas systems There are three types of CRISPR/Cas systems which all incorporate these RNAs and proteins known as "Cas" proteins (CRISPR associated). Types I and III both have Cas endonucleases that process the pre-crRNAs, that, when fully processed into crRNAs, assemble a multi-Cas protein complex that is capable of cleaving nucleic acids that are complementary to the crRNA.
• crRNAs are produced using a different mechanism where a trans-activating RNA (tracrRNA) complementary to repeat sequences in the pre-crRNA, triggers processing by a double strand- specific RNase III in the presence of the Cas9 protein.
• Cas9 is then able to cleave a target DNA that is complementary to the mature crRNA however cleavage by Cas 9 is dependent both upon base-pairing between the crRNA and the target DNA, and on the presence of a short motif in the crRNA referred to as the PAM sequence (protospacer adjacent motif).
• the tracrRNA must also be present as it base pairs with the crRNA at its 3' end, and this association triggers Cas9 activity.
• the Cas9 protein has at least two nuclease domains: one nuclease domain is similar to a HNH endonuclease, while the other resembles a Ruv endonuclease domain.
• the HNH-type domain appears to be responsible for cleaving the DNA strand that is
• sgRNA single-guide RNA
• the engineered tracrRNA: crRNA fusion, or the sgRNA guides Cas9 to cleave the target DNA when a double strand RNA:DNA heterodimer forms between the Cas associated RNAs and the target DNA.
• This system comprising the Cas9 protein and an engineered sgRNA containing a PAM sequence has been used for RNA guided genome editing (see Ramalingam ibid) and has been useful for zebrafish embryo genomic editing in vivo (see Hwang et al (2013) Nature
• nucleases can also be engineered to insert a peptide fusion inhibitor on to an HIV receptor to prevent HIV infection of T cells (see co-owned US patent publication no. 20120093787), where an example of such a peptide fusion inhibitor is C34 or fuzeon.
• HIV can be treated by using engineered nucleases to insert anti-HIV transgenes in safe harbor loci within the cell to combat the virus.
• anti-HIV genes may be selected from the group consisting of a sequence encoding a zinc finger transcription factor that represses an HIV polyprotein, a sequence encoding a zinc finger transcription factor that represses expression of an HIV receptor, a CCR5 ribozyme, an siRNA sequence targeted to an HIV polyprotein, a sequence encoding a Trim5alpha restriction factor, a sequence encoding an APOBEC3G restriction factor, a sequence encoding a RevMlO protein, a sequence encoding C46, other anti-HIV genes, a suicide cassette and combinations thereof.
• compositions of the invention may be used to treat or prevent HIV with a CRISPR/Cas system where the single guide RNA comprises sequences to target the CCR5 or CXCR4 gene for integration of a suitable anti-HIV transgene.
• the genome editing can include cleavage with site- specific nucleases for targeted insertion into a chosen genomic locus (see, e.g., co-owned U.S. Pat. No. 7,888,121). Nucleases specific for targeted genes can be utilized such that the transgene construct is inserted by either homology directed repair (HDR) or by end capture during non-homologous end joining (NHEJ) driven processes.
• Targeted loci include "safe harbor" loci such as the AAVS1, HPRT and CCR5 genes in human cells, and Rosa26 in murine cells (see, e.g., U.S. Pat. Nos.
• Nuclease-mediated integration offers the prospect of improved transgene expression, increased safety and expressional durability, as compared to classic integration approaches that rely on random integration of the transgene, since it allows exact transgene positioning for a minimal risk of gene silencing or activation of nearby oncogenes.
• Genome editing can also include the knocking out of genes in addition to insertion methods described above.
• a cell with a cleaved genome will resort to the error prone NHEJ pathway to heal the break. This process often adds or deletes nucleotides during the repair process ("indels") which may lead to the introduction of missense or non-sense mutations at the target site.
• CCR5-specific zinc finger nucleases are being used in Phase I/II trials to create a non-functional CCR5 receptor in T-cells, and thus prevent HIV infection (see U.S. Pat. No. 7,951,925). These cells are then re-introduced into the patient to treat HIV.
• the methods and compositions of the invention may be used to disrupt CCR5 alleles with a CRISPR/Cas system where the single guide RNA comprises sequences to target a human CCR5 gene (chr3:46411633-46417697), especially at or near the exon region
• One especially preferred region for targeting the CCR5 gene for knock out is the region near the delta-32 mutation region (at or near chr3:46414923-46415020). Another especially preferred region is around the chr3: 46414522-46414643, which encodes part of the second extracellular loop of the CCR5 protein.
• the region at or near the ATG protein translation initiation site is also especially preferred for genome modification, such as fusion of a C34 peptide to the N-terminus of CCR5 by targeted integration for anti-HIV therapy.
• One preferred region for targeting the CXCR4 gene for knock out is the region at or near chr2: 136872863- 136872982 that is an analog to the delta-32 mutation region in CCR5 gene.
• the region at or near chr2: 136875540- 136875687 near the ATG protein translation initiation site of exonl is especially preferred, and the region at or near chr2: 136873389- 136873558 near the splicing site of exon2 is especially preferred for gene modification, such as fusion of a C34 peptide to the N-terminus of CXCR4 by targeted integration for anti-HIV therapy.
• a sgRNA can be designed to bind to sequences anywhere in the CCR5 or CXCR4 locus, including, but not limited to, a sequence in one or more of these preferred targeting regions.
• nucleases, polynucleotides encoding these nucleases, donor polynucleotides and compositions comprising the proteins and/or polynucleotides described herein may be delivered ex vivo to the CD4/CD8 T cells and/or CD4/CD8 T cells having a
• CD45RA mt CD45RO mt phenotype by any suitable means.
• Nucleases and/or donor constructs as described herein may also be delivered using vectors containing sequences encoding one or more of the CRISPR/Cas system(s).
• vector systems including, but not limited to, plasmid vectors, DNA minicircles, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors;
• herpesvirus vectors and adeno-associated virus vectors etc., and combinations thereof. See, also, U.S. Pat. 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. Publication No. 20140335063, incorporated by reference herein in their entireties.
• any of these vectors may comprise one or more of the sequences needed for treatment.
• the nucleases and/or donor polynucleotide may be carried on the same vector or on different vectors.
• each vector may comprise a sequence encoding one or multiple nucleases and/or donor constructs.
• Non-viral vector delivery systems include DNA plasmids, DNA minicircles, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
• Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
• Methods of non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, naked RNA, capped RNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids.
• 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 for example U.S. Pat. No. 6,008,336).
• Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam.TM. and Lipofectin.TM.).
• Cationic and neutral lipids that are suitable for efficient receptor- recognition lipofection of polynucleotides include those of Feigner, WO 91/17424, WO 91/16024.
• lipidmucleic acid complexes including targeted liposomes such as immunolipid complexes
• Boese 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. Pat. 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).
• Additional methods of delivery include the use of packaging the nucleic acids to be delivered into EnGeneIC delivery vehicles (ED Vs). These ED Vs are specifically delivered to target tissues using bispecific antibodies where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV. The antibody brings the EDVs to the target cell surface and then the EDV is brought into the cell by endocytosis. Once in the cell, the contents are released (see MacDiarmid et al. (2009) Nature
• RNA or DNA viral based systems for the delivery of nucleic acids encoding engineered CRISPR/Cas systems take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
• Viral vectors can be administered directly to subjects ⁇ in vivo ) or they can be used to treat cells in vitro and the modified cells are administered to subjects ⁇ ex vivo).
• Conventional viral based systems for the delivery of CRISPR/Cas systems include, but are not limited to, retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
• Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system depends on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
• Widely used retroviral vectors include those based upon murine leukemia vims (MuLV), gibbon ape leukemia vims (GaLV), Simian Immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et ah, J. Virol. 66:2731-2739 (1992); Johann et ah, 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/US 94/05700).
• MiLV murine leukemia vims
• GaLV gibbon ape leukemia vims
• SIV Simian Immunodeficiency virus
• HAV human immunodeficiency virus
• Adenoviral based systems can be used.
• Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.
• Adeno-associated virus (“AAV”) vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No.
• At least six viral vector approaches are currently available for gene transfer in clinical trials, which utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent.
• pLASN and MFG-S are examples of retroviral vectors that have been 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 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al., Immunol Immunother. 44(1): 10-20 (1997); Dranoff et al., Hum. Gene Ther. 1:111-2 (1997).
• Recombinant adeno-associated virus vectors are a promising alternative gene delivery systems based on the defective and nonpathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from a plasmid that retains 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 genomes of the transduced cell are key features for 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, and all variants thereof, can also be used in accordance with the present invention.
• Ad Replication-deficient recombinant adenoviral vectors
• Ad can be produced at high titer and readily infect a number of different cell types.
• Most adenovirus vectors are engineered such that a transgene replaces the Ad El a, Elb, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans.
• Ad vectors can transduce multiple types of tissues in vivo , including non dividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity.
• Ad vector An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for anti-tumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al., Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).
• Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and .psi.2 cells or PA317 cells, which package retrovirus.
• Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line.
• AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome.
• ITR inverted terminal repeat
• Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
• the cell line is also infected with adenovirus as a helper.
• the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
• the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
• Gene editing vectors can be delivered ex vivo to CD4/CD8 T cells and/or CD4/CD8 T cells having the CD45RA int CD45RO int phenotype followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
• Formulations including the gene editing vectors for ex vivo administration can include suspensions in liquid or emulsified liquids.
• the active ingredients often are mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof.
• the formulation may contain minor amounts of auxiliary substances, such as, wetting or emulsifying agents, pH buffering agents, stabilizing agents or other reagents that enhance the effectiveness of the pharmaceutical composition.
• the selected, enriched population CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype produced by the methods described herein 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.
• the carrier can be any of those conventionally used for the administration of cells.
• Such pharmaceutically acceptable carriers are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which has no detrimental side effects or toxicity under the conditions of use.
• compositions can be prepared in unit dosage forms for administration to a subject.
• the amount and timing of administration are at the discretion of the treating clinician to achieve the desired outcome.
• the compositions can be formulated for systemic (such as intravenous) or local (such as intra-tumor) administration.
• an enriched population of CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA mt CD45RO mt phenotype is formulated for parenteral administration, such as intravenous administration.
• compositions including enriched population of CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype as disclosed herein can be used, for example, for the treatment of HIV in a subject.
• compositions for administration can include a solution of the enriched population of CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA mt CD45RO mt phenotype provided in a pharmaceutically acceptable carrier, such as an aqueous carrier.
• a pharmaceutically acceptable carrier such as an aqueous carrier.
• aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
• These compositions may be sterilized by conventional, well known sterilization techniques.
• compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, adjuvant agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
• auxiliary substances such as pH adjusting and buffering agents, toxicity adjusting agents, adjuvant agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
• CD45RA mt CD45RO mt phenotype in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject's needs. Actual methods of preparing such dosage forms for use in in gene therapy, immunotherapy and/or cell therapy are known, or will be apparent, to those skilled in the art.
• the enriched population of CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype can be added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of from 0.5 to 15 mg/kg of body weight.
• An enriched population of CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level.
• the dose e.g., number of the CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA mt CD45RO mt phenotype administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject or animal over a reasonable time frame.
• the number of the CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype should be sufficient to treat HIV over a period of from about 6 months, 1 year, 2 years, 3 years, 4 years or more from the time of
• the number of the CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype will be determined by, e.g., the efficacy of the CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.
• the number of the of CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of an enriched population of CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the
• CD45RA mt CD45RO mt phenotype typically, the attending physician will decide the number of the inventive CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the
• CD45RA mt CD45RO mt phenotype with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, route of administration, and the severity of the condition being treated.
• factors such as age, body weight, general health, diet, sex, route of administration, and the severity of the condition being treated.
• CD4/CD8 T cells having the CD45RA int CD45RO int phenotype can be about 10 x 10 4 to about 10 x 10 11 cells per infusion, about 10 x 10 5 cells to about 10 x 10 9 cells per infusion, or 10 x 10 7 to about 10 x 10 9 cells per infusion.
• the CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype can be used in methods of treating or preventing HIV infection in a subject in need thereof.
• a method of treating or preventing HIV infection in a subject can include administering to the subject an enriched population of CCR5 and/or CXCR4 gene edited CD4/CD8 T cells can have a CD45RA int CD45RO int phenotype described herein in an amount effective to treat or prevent HIV in a subject.
• administration of the composition or enriched T-cell population to a subject with HIV is capable of promoting at least one of a sustained increase in absolute CD4 cell numbers, restoration of HIV specific T cell immunity, and a substantial decay in HIV reservoir in the subject.
• the subject has undergone and/or continues to undergo antiretroviral therapy
• the administered CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype can be cells that are allogeneic or autologous to the host or subject.
• the cells are autologous to the subject.
• the CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype can be administered in combination with an activator of latent HIV expression.
• activators of latent HIV expression can be used in the compositions and methods described herein.
• an activator of latent HIV expression can include, but is not limited to, histone deacetylase (HD AC) inhibitors and protein kinase C agonists.
• HD AC histone deacetylase
• HD AC inhibitors induce the transcriptional activation of the HIV-1 promoter.
• An HD AC inhibitor may be any molecule that effects a reduction in the activity of a histone deacetylase. This includes proteins, peptides, DNA molecules (including antisense), RNA molecules (including iRNA agents and antisense) and small molecules.
• a HD AC inhibitor is a small interfering RNA (siRNA), for example, a si/shRNA directed against HDAC1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
• siRNA small interfering RNA
• HDAC inhibitors include any salts, crystal structures, amorphous structures, hydrates, derivatives, metabolites, stereoisomers, structural isomers, and prodrugs of the HDAC inhibitors described herein.
• an HDAC inhibitor can include short-chain fatty acids (e.g., Sodium Butyrate, Isovalerate, Valerate, 4-Phenylbutyrate (4-PBA), Phenylbutyrate (PB), Propionate, Butyramide , Isobutyramide, Phenylacetate, 3-Bromopropionate, Tributyrin, Valproic acid (Vpa), Valproate, Valproate semisodium and pivaloyloxymethyl butyrate (PIVANEX)).
• short-chain fatty acids e.g., Sodium Butyrate, Isovalerate, Valerate, 4-Phenylbutyrate (4-PBA), Phenylbutyrate (PB), Propionate, Butyramide , Isobutyramide, Phenylacetate, 3-Bromopropionate, Tributyrin, Valproic acid (Vpa), Valproate, Valproate semisodium and pivaloyloxymethyl buty
• an HDAC inhibitor can include a hydroxamic acid derivative (e.g., suberoylanilide hydroxamic acid (SAHA, vorinostat), Trichostatin analogs such as Trichostatin A (TSA) and Trichostatin C, m-Carboxycinnamic acid bishydroxamide (CBHA), Pyroxamide, Salicylbishydroxamic acid, Suberoyl bishydroxamic acid (SBHA), Azelaic bishydroxamic acid (ABHA) Azelaic-l-hydroxamate-9-anilide (AAHA), 6-(3- Chlorophenylureido) carpoic hydroxamic acid (3C1— UCHA), Oxamflatin [(2E)-5-[3- [(phenylsulfonyl) amino]phenyl]-pent-2-en-4-ynohydroxamic acid], A- 161906 Scriptaid, PX
• an HDAC inhibitor can include benzamide derivatives (e.g., CI-994; MS-275 [N-(2-aminophenyl)-4-[N-(pyridin-3-yl
• an HD AC inhibitor can include cyclic peptides (e.g ., Trapoxin A (TPX)-cyclic tetrapeptide (cyclo-(L-phenylalanyl-L-phenylalanyl-D- pipecolinyl-L-2-amino-8-oxo-9, 10-epoxy decanoyl)), FR901228 (FK 228, depsipeptide), FR225497 cyclic tetrapeptide, Apicidin cyclic tetrapeptide [cyclo(N— O-methyl-L- tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)], Apicidin la, Apicidin lb, Apicidin Ic, Apicidin Ila
• Additional HD AC inhibitors can include natural products, such as psammaplins and Depudecin, Electrophilic ketone derivatives such as Trifluoromethyl ketones, a-keto amides such as N-methyl-a-ketoamides, LSD1 polypeptide, TNF-alpha (TNFa), an inducible transcription factor NF-AT (nuclear factor of activated T cells), and Anti- IkBa or IkBe agents.
• natural products such as psammaplins and Depudecin
• Electrophilic ketone derivatives such as Trifluoromethyl ketones
• a-keto amides such as N-methyl-a-ketoamides
• LSD1 polypeptide such as TNF-alpha (TNFa)
• TNF-alpha (TNFa) TNF-alpha (TNFa)
• NF-AT inducible transcription factor of activated T cells
• Anti- IkBa or IkBe agents can include natural products, such
• CD45RA mt CD45RO mt phenotype alone or in combination with the activators of latent HIV expression described herein can be administered to a subject that is latently infected with HIV, e.g., a human latently infected with HIV.
• the subject can include a subject having a persistent HIV reservoir despite treatment with antiretroviral therapy (e.g., HAART).
• the therapeutically effective amount is the amount of the CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype to significantly decrease a latent HIV reservoir in a latently HIV infected subject.
• a therapeutically effective amount of CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype, and optionally an activator of latent HIV expression can be administered to the subject in combination with another therapeutic agent, which useful in the treatment of HIV infection, such as a component used for HAART or immunotoxins.
• the CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype described herein may be combined with one or more additional therapeutic agents useful in the treatment of HIV infection.
• additional therapeutic agents useful in the treatment of HIV infection may be combined with one or more additional therapeutic agents useful in the treatment of HIV infection.
• the scope of combinations of the CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype with HIV/AIDS antivirals, immunomodulators, anti-infectives or vaccines is not limited to the following list, and includes in principle any combination with any pharmaceutical composition useful for the treatment of AIDS.
• the HIV/AIDS antivirals and other agents will typically be employed in these combinations in their conventional dosage ranges and regimens as reported in the art.
• antiviral agents include (but not restricted) ANTIVIRALS
• 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 infections HIV infections, AIDS, ARC (nucleoside reverse transcriptase inhibitor); acyclovir Burroughs Wellcome HIV infection, AIDS, ARC, 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 GS 840 (RTI); AL-721 Ethigen ARC, PGL, HIV positive, (Los Angeles, Calif.), AIDS alpha interferon GlaxoSmithKline Kaposi's sarcoma, HIV, in combination w/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 LM 427 (Dublin, Ohio) Er
• ARC dextran Sulfate Ueno Fine Chem. Ind. AIDS, ARC, HIV Ltd. (Osaka, Japan) positive asymptomatic ddC Hoffman-La Roche HIV infection, AIDS, ARC (zalcitabine, (HIVID) (nRTI); dideoxycytidine ddl Bristol-Myers Squibb HIV infection, AIDS, ARC; Dideoxyinosine (VIDEX) combination with AZT/d4T (nRTI) DPC 681 & DPC 684 DuPont HIV infection, AIDS, ARC (PI) DPC 961 & DPC 083 DuPont HIV infection AIDS, ARC (nnRTRI); emvirine Triangle Pharmaceuticals HIV infection, AIDS, ARC (COACTINON) (non-nucleoside reverse transcriptase inhibitor); ELIO Elan Corp, PLC HIV infection (Gainesville, Ga.) efavirenz DuPont HIV infection,
• HIV infection HIV infection, AIDS, ARC recombinant human; Triton Biosciences AIDS, Kaposi's sarcoma, interferon beta (Almeda, Calif.); ARC interferon alfa-n3 Interferon Sciences ARC, AIDS indinavir; Merck (CRIXIVAN) HIV infection, AIDS, ARC, asymptomatic HIV positive, also in combination with AZT/ddEddC (PI); ISIS 2922 ISIS Pharmaceuticals CMV retinitis JE2147/AG1776; Agouron HIV infection, AIDS, ARC (PI); KNI-272 Nat'l Cancer Institute HIV-assoc.
• nevirapine Boeheringer HIV infection AIDS, Ingleheim ARC (nnRTI); (VIRAMUNE) novapren Novaferon Labs, Inc. HIV inhibitor (Akron, Ohio); pentafusaide Trimeris HIV infection, AIDS, ARC T-20 (fusion inhibitor); peptide T Peninsula Labs AIDS octapeptide (Belmont, Calif.) sequence
• PRO 542 Progenies HIV infection, AIDS, ARC (attachment inhibitor); PRO 140 Progenies HIV infection, AIDS, ARC (CCR5 co-receptor inhibitor); trisodium Astra Pharm.
• VREAD HIV infection
• AIDS HIV infection
• ARC ARC
• tipranavir PNU-140690
• the additional therapeutic agent may be used individually, sequentially, or in combination with the CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA mt CD45RO mt phenotype.
• Administration to a subject may be by the same or different route of administration or together in the same pharmaceutical formulation.
• the CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype and an activator of latent HIV expression may be coadministered with any HAART regimen or component thereof.
• the current standard of care using HAART is usually a combination of at least three nucleoside reverse transcriptase inhibitors and frequently includes a protease inhibitor, or alternatively a non nucleoside reverse transcriptase inhibitor. Subjects who have low CD4 + cell counts or high plasma RNA levels may require more aggressive HAART.
• the CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA mt CD45RO mt phenotype and, optionally, an activator of latent HIV expression may be coadministered to the subject with a“cocktail” of nucleoside reverse transcriptase inhibitors, non-nucleoside HIV reverse transcriptase inhibitors, and protease inhibitors.
• the CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA mt CD45RO mt phenotype and an HD AC inhibitor may be coadministered with a cocktail of two nucleoside reverse transcriptase inhibitors (e.g ., ZIDOVUDINE (AZT) and LAMIVUDINE (3TQ), and one protease inhibitor (e.g., INDINAVIR (MK-639)).
• a cocktail of two nucleoside reverse transcriptase inhibitors e.g ., ZIDOVUDINE (AZT) and LAMIVUDINE (3TQ)
• one protease inhibitor e.g., INDINAVIR (MK-639)
• the CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype and optionally an activator of latent HIV expression, such as an HD AC inhibitor may also be coadministered to the 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)).
• the CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the CD45RA int CD45RO int phenotype and optionally an HD AC inhibitor may be coadministered to the subject with a cocktail of one nucleoside reverse transcriptase inhibitor (e.g ., STAVUDINE (d4T)), one non-nucleoside
• nucleoside reverse transcriptase inhibitor e.g., ZIDOVUDINE (AZT)
• ZIDOVUDINE ZIDOVUDINE
• protease inhibitors e.g., NELFINAVIR (AG- 1343) and SAQINAVIR (Ro-31-8959)
• Coadministration in the context of this invention is defined to mean the administration of more than one therapeutic agent in the course of a coordinated treatment to achieve an improved clinical outcome. Such coadministration may also be coextensive, that is, occurring during overlapping periods of time.
• immunotoxins can be coadministrered to a subject with the CCR5 and/or CXCR4 gene edited CD4/CD8 T cells having the
• an immunotoxin is an immunotoxin targeted to an HIV protein expressed on the exterior of cells, such as the viral envelope glycoprotein or a portion thereof.
• the term“immunotoxin” refers to a covalent or non- covalent linkage of a toxin to an antibody, such as an anti HIV envelope glycoprotein antibody.
• the toxin may be linked directly to the antibody, or indirectly through, for example, a linker molecule.
• the toxin can be selected from the group consisting of ricin-A and abrin-A.
• Activation 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, e.g., production of infectious particles, reverse transcriptase activity, secreted antigens, cell- surface antigens, soluble antigens, HIV RNA and HIV DNA, etc.
• the methods described herein may optionally include the step of determining or detecting activation of latent HIV expression. In one embodiment, such a method comprises determining or detecting an mRNA, e.g., an HIV mRNA.
• mRNAs such as Tat mRNA, NF-KB mRNA, NF-AT mRNA and other mRNAs encoding polypeptides can also be determined using the well known methods including but not limited to hybridization and amplification based assays.
• amplification-based assays are used to measure the expression level of an HIV gene.
• activation of latent HIV expression can be detecting by determining the expression level of an HIV polypeptide.
• the expression level of an HIV polypeptide may be determined by several methods, including, but not limited to, affinity capture, mass spectrometry, traditional immunoassays directed to HIV proteins (such as gpl20 and reverse transcriptase), PAGE, Western Blotting, or HPLC as further described herein or as known by one of skill in the art.
• Detection paradigms that can be employed to this end include optical methods, electrochemical methods (voltametry and amperometry techniques), atomic force
• radio frequency methods e.g., multipolar resonance spectroscopy.
• optical methods in addition to microscopy, both confocal and non-confocal, are detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or
• global sequencing and 454 pyrosequensing of HIV based vector constructs and the PCR products described herein can be performed to confirm the production and purity of an autologous vims population.
• 454 is a simple, efficient, and cost effective means to obtain approximate genetic diversity in the samples.
• DNA vectors and plasma RNA will be amplified with bar-coded primers and then sequenced using a 454 JR to obtain an average of -2000 reads per amplicon/s ample.
• the SB-728-0902 clinical trial is a Phase 1, open label, uncontrolled, nonrandomized study of individuals with chronic HIV infection treated with ART
• the SB-728-1101 clinical trial is a Phase 1, open label, uncontrolled, nonrandomized study of individuals with chronic HIV infection treated with ART (ClinicalTrials.gov #NCT01543152).
• the study was sponsored by Sangamo Therapeutics and was conducted at 12 centers in the United States between March 2012 and January 2017.
• the primary objective of the study was to evaluate the safety and tolerability of escalating doses of cyclophosphamide (CTX) pre-treatment to promote CD4+ T cell expansion after administration of a single dose of SB-728-T cells.
• CTX cyclophosphamide
• ART was reinstituted in participants whose CD4+ T cell counts dropped to ⁇ 500 cells/pL and/or whose HIV-RNA increased to >100,000 copies/mL on three consecutive weekly measurements. All participants completed the 1-year study and were enrolled in a 3- year long-term safety study with the exception of one participant who withdrew from the study. One participant (03-003) did not interrupt ART.
• Eligible participants were 18 years of age or older and were infected with HIV, as documented by ELISA. Participants were aviremic (undetectable HIV RNA), receiving stable ART with CD4+ T cell counts between 200 and 500 cell/pL, , had adequate venous access and no contraindications to leukapheresis.
• the key exclusion criteria included a SNP at the CCR5 zinc finger nuclease target region, current or prior AIDS diagnosis, receiving therapy with maraviroc or immunosuppressives, and hepatitis B or hepatitis C co-infection. SB-728- 1101 Trial
• Eligible participants were 18 years of age or older and were infected with HIV, as documented by ELISA. Participants were aviremic on stable ART with CD4+ T cell counts >500/pL, had R5 tropic HIV, and willing to discontinue current ART during the treatment interruption.
• the key exclusion criteria included adenoviral neutralizing antibodies >40, a SNP at the CCR5 zinc finger nuclease target region, current or prior AIDS diagnosis, receiving therapy with maraviroc or immunosuppressives, and hepatitis B or hepatitis C co- infection.
• SB-728-T refers to autologous CD4+ enriched T cells that have been transduced ex vivo with SB-728, a replication deficient recombinant Ad5/35 viral vector encoding the CCR5 specific ZFNs (SBS8196z and SBS8267), and includes a mixture of gene edited and un-edited cells.
• CCR5- specific ZFNs induces a double stranded break in the cell’s DNA which is repaired by cellular machinery leading to random sequence insertions or deletions (indels) in -25% of transduced cells. These indels disrupt the CCR5 coding sequence leading to frameshift mutation and termination of protein expression.
• PBMCs peripheral blood mononuclear cells
• cryopreserved samples at different time points varied between participants; consequently, time points were grouped into early (14-28 days), mid (4-7 months or 9-10 months), late (11-12 months), and long term (2-3 or 3-4 years) post infusion time points.
• Baseline samples included cryopreserved PBMCs from the initial leukapheresis (2-3 months before infusion) as well as from a small volume blood draw 1-2 weeks before infusion.
• PBMCs from participants 1-01, 1-02, and 1-03 were not cryopreserved until months 6 or 8 post infusion.
• cryopreserved PBMCs from the initial leukapheresis (2-3 months before infusion) as well as from a small volume blood drawn 1-2 weeks before infusion.
• Manufacturing samples (SB- 728-T products) were also available for Cohorts 3-5 participants.
• Rectal biopsies were performed for participants of the SB-928-0902 trial at baseline, day 14, month 3, 6 and 12 (n varied between 3 and 9 participants per time point).
• Mucosal mononuclear cells were isolated from sigmoid colon biopsies obtained by endoscopy via a combination of collagenase digestion and teasing with 18G needles.
• Inguinal lymph nodes were biopsied from 3 volunteers at one time point (between 9 and 18 months post SB-728-T infusion). Tissues were processed into single cells as described in Anton et al 47 and genomic DNA were isolated for assessment of CCR5 gene modifications.
• ZFN-mediated gene modification can generate a wide range of frame-shift mutations to disrupt the CCR5 gene locus.
• a PCR-based assay was developed to measure the acquisition of a unique duplication of 5-nucleotide (Pentamer) DNA sequence, CTGAT, at the ZFN cleavage site in approximately 25% of the gene edited alleles.
• Genomic DNA gDNA was extracted from PBMCs using a commercially available kit (Masterpure DNA Purification kit, Epicenter, Madison, WI).
• a standard PCR was performed with 5pg of gDNA to amplify a 1.1 kb region that contains CCR5 gene modifications.
• This 1.1 kb amplicon is subsequently evaluated with the two independent qPCRs, one specific for the Pentamer Duplication- CCR5 gene edited allele (by using a primer that contains the Pentamer Duplication), and a second that amplifies all CCR5 alleles.
• Duplication-specific templates and the total number of CCR5 alleles yields Pentamer Duplications per 1 million PBMCs.
• the assay has a sensitivity of one CCR5 gene edited allele per 10 5 total CCR5 alleles.
• the frequency of CCR5 gene edited cells in PBMCs was estimated by multiplying the frequency of Pentamer Duplication gene edited cells by 4.
• the Cel-I nuclease specifically cleaves DNA duplexes at the sites of distortions created by either bulges or mismatches in the double helical DNA structure.
• the locus of interest (ZFN binding sites in CCR5) was PCR amplified from genomic DNA, and the levels of modification at each locus were determined by paired-end deep sequencing on an Illumina MiSeq sequencer. Paired sequences were merged via SeqPrep (John St. John, https://github.com/jstjohn/SeqPrep, unpublished). A Needleman- Wunsch alignment was performed between the target amplicon genomic region and the obtained Illumina sequence to map indels.
• CCR5 gene edited memory subset cell counts were estimated by multiplying each memory subset cell counts by the frequency of CCR5 gene edited alleles within each memory subset as determined by CCR5 sequencing.
• the level of CCR5 gene edited alleles persisting in a participant relative to the amount of CCR5 gene edited cells infused can be estimated using the measured values of CCR5 modification by the Pentamer Duplication marker and CD4+ cell count with the assumptions; 1) blood volume is 4.7 liters, 2) approximately 2.5% of all CD4+ T cells are found in the periphery 49 and 3) SB-728-T products distribution is similar to endogenous CD4+ T cells (levels of CCR5 modification in CD4+ T cells from the sigmoid and inguinal nodes are similar to that in the periphery, Fig. 8E).
• PerCPe710 (clone UCHL1) (Biolegend), and aqua fluorescent reactive dye (a dead cell marker) (Invitrogen).
• the TSCM panel included CD95 PE-Cy7 (clone DX2), CD58 PE (clone 1C3), CD 127 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).
• CCR7 PE-CF594 (clone 150503), CTLA-4 APC (clone BNI3), CD31 PE (clone WM59) (BD Biosciences), Tim-3 BV421 (clone F38-2E2), PD-1 PE-Cy7 (clone EH12.2H7) (Biolegend), and LAG-3 FITC (clone 17B4) (Novus Biologicals).
• a minimum of 100,000 live cells were acquired within 24hrs using a BD LSR-II and analyzed using FlowJo version 9.
• CD4+ T cells were first isolated from PBMCs by negative magnetic selection (StemCell), and then surface stained with 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 0323) (eBioscience), CD8 PerCP (clone SKI), CD45RA BV 650 (clone HI100), CD45RO PerCPe710 (clone UCHL1), CD19 BV 510 (clone H1B19) (Biolegend), CCR
• CD4+ T cells Up to 200,000 total CD4+ T cells as well as CD4+ T cell subsets were then sorted with the FACSAria (Becton Dickinson) and stored as dry pellets at -80°C until analysis.
• FACSAria Becton Dickinson
• 10,000 sorted cells were collected directly into RNAse-free 1.5mL eppendorf tubes containing 500pL of RLT buffer with 1% b-mercaptoethanol and stored at -80°C until analysis.
• gDNA genomic DNA
• PCR droplets were prepared according to manufacturer's recommendations. Briefly, a 20pL of multiplex PCR mixture is prepared by mixing 250 or 500 ng of the digested gDNA with the ddPCRTM 2x Master Mix and two Taqman primer/probe sets. PCR droplets were generated in a DG8TMcartridge using the QX-100 droplet generator, where each 20pL PCR mixture was partitioned into
• PCR droplets were transferred into a 96-well PCR plate and sealed with foil. Standard PCR was performed with a Bio-Rad C1000
• HIV DNA copy number was evaluated using the QX-100 Droplet Digital PCR system (Bio- Rad, Hercules, CA). The PCR-positive and PCR-negative droplets for HIV gag and RPP30 were determined and template concentrations were calculated by Poisson analysis. HIV copy number was determined by normalizing HIV gag concentration to RPP30 concentration.
• HIV Tropism was evaluated using the commercial Trofile® DNA assay (Monogram BioSciences/ LabCorp, South San Francisco, CA). Viral envelope DNA sequence was extracted from PBMCs. HIV tropism is determined using a cell based transduction assay where HIV env protein sequences are amplified from PBMC samples, subcloned as a library, packaged into lentiviral vectors, and evaluated using co-receptor restricted cell lines.
• PBMCs were rested for 12 hours prior to stimulation of 2 million cells each with Brefeldin A (5pg/mL) (Sigma Aldrich) and either gag peptides (lpg/peptide/mL; NIH AIDS reagent program), Staphylococcal enterotoxin B (SEB; lpg/mL) or complete media (mock) for 6 hours.
• Brefeldin A 5pg/mL
• gag lpg/peptide/mL lpg/peptide/mL
• SEB Staphylococcal enterotoxin B
• complete media mock
• 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 H1B 19) (Biolegend), and aqua fluorescent reactive dye (Invitrogen), permeabilised with 0.05% Saponin and stained intracellularly with IL-2 PerCP-Cy5.5 (clone MQ1-17H12), IFNy APC (clone B27) and TNFa Alexa Fluor 488 (clone MAB 11) (BD Biosciences) prior to fixation with 2% formaldehyde.
• CD3 Alexa 700 (clone UCHT1), CD8 Pacific Blue (clone RPA
• Cells were acquired within 24 hours using a BD LSR-II. A minimum of 500,000 live events was acquired. Cells were analyzed using FlowJo version 9, and the Boolean gating function was used to determine the distribution of polyfunctional CD8+ T subsets.
• TCR T cell Receptor
• TCR repertoire analysis was performed with the immunoSEQ assay (Adaptive Biotechnologies, Seattle, WA).
• the immunoSEQ method amplifies rearranged TCR CDR3 sequences by multiplex PCR to explore all nb and Ib combinations from isolated genomic DNA, and uses high-throughput sequencing technology to sequence TCR CDR3 chains to determine the composition of various T cell clones within each sample.
• TCR diversity is assessed using the Shannon entropy index, which accounts for both the number of unique clones (richness) and clone distribution (evenness) of the TCR nb CDR3 sequences present in each sample.
• a larger Shannon entropy index reflects a more diverse distribution of the TCR nb CDR3 sequences.
• CD4+ or CD8+ subsets were sorted into RLT buffer as described above. Specifically, CD4+ TCM, CD4+ TTM, CD4+ TEM and CD8+ total memory cells were sorted at baseline and month 12. In addition, CD4+ memory subsets were also sorted at year 3-4 and included CD45RA mt RO mt TSCM, TCM, and TEM cells. Sorted cells were lysed for RNA extraction as per manufacturer's instructions (Qiagen, Valencia, CA). T7 oligo(dT) primed reverse transcription reactions were performed followed by in vitro transcription.
• the difference in gene expression level between the different time points or subsets was determined by performing longitudinal donor-paired analysis.
• the moderated t-test implemented in the LIMMA package was used to assess the statistical significance ( P ⁇ 0.05) of differential expression of genes between baseline and month. All microarray data have been deposited in GEO under accession number GSE66214.
• GSEA Gene Set Enrichment Analysis
• CD45RA int RO int TSCM in year 3-4 samples that correlated with the frequency of total HIV DNA copy per 10 6 PBMCs at year 2-4 and CD45RA int RO int TSCM counts at year 3-4.
• GSEA GSEA
• the paired Wilcoxon rank-sum two-tailed test was used to perform non- parametric donor-paired two-sided analysis of post infusion changes in CD4+ total T cell and subset counts, CD4:CD8 ratio, T cell function, immune checkpoint blockers, and integrated HIV DNA compared to baseline.
• the Wilcoxon rank-sum two-tailed test was also used to compare the levels of CCR5 gene edited alleles between subsets and to compare the diversity of the TCR repertoire and CCR5 gene edited alleles between SB-738-T products and long term time points.
• the Mann-Whitney two-tailed test was used to performed unpaired non- parametric two-sided comparisons in instances where the number of matched participants varied across time points and contained less than 6 matched pairs at a given time point, such as for the frequencies of CD95+ cells post infusion compared to baseline, and the levels of CCR5 gene edited alleles between the different CD4+ memory subsets in the SB-728-0902 study (Pentamer Duplication and CCR5 DNA sequencing).
• the Spearman’s rho (p) test was used to perform non-parametric correlation analysis between various measures and clinical outcomes, including delta CD4+ T cell counts (SB-728-0902), changes in the size of the reservoir calculated using the ratio of the last measured values (year 2-4 time points) over baseline (SB-728-0902), and control of viral replication (SB-728- 1101).
• SB-728-0902 delta CD4+ T cell counts
• SB-728-0902 changes in the size of the reservoir calculated using the ratio of the last measured values (year 2-4 time points) over baseline
• SB-728- 1101 control of viral replication
• AIC Akiake Information Criterion
• BIC Bayesian Information Criterion
• CD45RA int RO int (TSCM2,TSCM2GE), stem memory CD45RA + (TSCM1, TSCM IGE), central memory (CM, CM GE ), transitional memory (TM, TM GE ) and effector memory (EM, EM GE ), where a GE subscript refers to the CCR5 gene-edited CD4 T-cells.
• TSCM2,TSCM2GE stem memory CD45RA +
• CM central memory
• TM transitional memory
• EM effector memory
• the HIV DNA decay post infusion in a participant from the SB-728-0902 study due to dilution by the amount of cells infused can be estimated using the measured values of CCR5 gene modification by the Pentamer Duplication marker and of CCR5 gene
• Participant 1-02 had elevated anti- adenovirus titers which may have impeded on the levels of engraftment of CCR5 gene edited cells and persistence of CD45RA int RO int TSCM cells, a population highly enriched in gene edited cells (SB-728-T products are derived from transduction with recombinant Ad5/F35 adenoviral vector encoding the CCR5 targeting ZFNs), and therefore was excluded from selective analyses focused on correlating the expansion of CCR5 gene edited cells and the CD45RA int RO int TSCM subset with HIV reservoir decay (such as in Fig. lc-d, Fig. 2d-e, and Fig. 3g-i).
• CCR5 gene edited CD4+ T cells were detected for up to 4 years in PBMCs (mean of 0.8% marked PBMCs and mean of 2.7% marked CD4+ T cells) and up to 12 months (last measured time point) in rectal biopsies and lymph nodes (Fig. lOd, lOe).
• a two-phase decay model was used to determine if the persistence of infused cells with low levels of integrated HIV DNA solely contributed to the decay of the HIV reservoir through dilution during the 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 of 30.47% (95% Cl, 9.664-51.28) of the decline was observed. After which the levels of HIV DNA continued to decrease at a slower rate with a half-life of 211 days (95% Cl; 56-365).
• a novel memory stem cell-like CD4+ T cell subset contributes to restoration of T cell homeostasis and correlates with reservoir decay
• CD45RA int RO int cells were present in the SB-728-T products, were highly enriched in CCR5 gene edited alleles and significantly increased in absolute numbers at every time point analyzed post infusion.
• the frequency of CD95+CD58+ cells (markers expressed in TSCM within the CD45RA int RO int and CD45RA + RO subsets correlated positively with expansion of CCR5 gene edited cells.
• Levels of the Pentamer Duplication marker (a sequence tag of CCR5 gene edited cells) were specifically enriched within CD45RA int RO int CD95+ cells (referred to as CD45RA int RO int TSCM) and CD45RA + RO CD95+ cells (referred to as CD45RA + TSCM) with approximately 14- and 21-fold higher levels in CD45RA mt RO mt TSCM compared to central memory (TCM) or transitional memory (TTM) cells at years 3-4; respectively (Fig. 3B).
• CD45RA int RO int TSCM CD45RA int RO int TSCM
• CD45RA + RO CD95+ cells referred to as CD45RA + TSCM
• TCM central memory
• TTM transitional memory
• TCM cells are defined as CD45RA-, CD45RO+, CCR97+, and CD27+
• TTM cells are defined as CD45RA-, CD45RO+, CCR79-, and CD27+
• CD45RAintROint TSCM are defined as CD45RAint, CD495ROint, CCR7+, CD27+, CD127+, CD28+, CD58+, and CD95 Results are shown only for significant correlations with P ⁇ 0.05 and false discovery rate (FDR) ⁇ 0.25
• CD45RA int RO int TSCM To investigate the role of CD45RA int RO int TSCM on the decay of the HIV reservoir, we first quantified levels of integrated HIV DNA in SB-728-T products and at year 3-4 samples in CD4+ T cell subsets and found that CD45RA int RO int TSCM cells had significantly lower levels of integrated HIV DNA compared to other memory subsets (1.99 loglO, 95% Cl: 1.64-2.34 in CD45RA mt RO mt TSCM at years 3-4 vs.
• CD45RA int RO int TSCM express genes associated with quiescence and self-renewal and can differentiate into other memory subsets
• GSEA Gene Set Enrichment Analysis
• CD45RA int RO int TSCM cells can differentiate into and replenish the pool of more differentiated memory cells.
• Our analysis placed the CD4+ CD45RA int RO int TSCM cells as less differentiated than TCM TTM, and TEM.
• CD45RA mt RO mt TSCM cells undifferentiated status of CD45RA mt RO mt TSCM cells was confirmed by analyzing the expression levels of transcription factors associated with Thl (T-bet and Eomes), Th2 (GATA-3) and Thl7 (RORgt) lineage commitment. Similar to naive cells and CD45RA + TSCM, CD45RA int RO int TSCM cells did not express Th-specific transcription factors (Fig. 3d). Further analysis of immune checkpoint markers showed that CD45RA mt RO mt CD95 + cells have significantly lower levels of PD-1, TIGIT, and SEAM than TTM and TEM suggesting that CD45RA int RO int CD95 + cells are less exhausted than other memory subsets (Fig. 28B).
• CD45RA int RO int TSCM exhibit stem cell properties that include longevity and multipotency and are precursors to the more differentiated than TCM, TEM and TEM memory cells.
• pro-inflammatory signatures in this subset could point to an increase in their ability to differentiate into more“effector” like cells.
• the combined leading-edge genes associated with the Wnt-signaling cascade were positively correlated with the long-term increases in CD4+ T cell counts and negatively correlated with the reduction of the HIV reservoir post infusion.
• ATI was extended in six individuals that showed viral load measurements below 10,000 copies/mL and CD4+ T cell counts above 500 cells/pl at week 22 who then spent 0.5-2 years on ATI. Viral load at month 12 for the 5 participants that remained on ATI at that time point ranged from 130 to 16,000 copies/mL.
• HLA human leukocyte antigen
• sensitivity analysis also showed that the proliferation rates of the CCR5 gene edited CD45RA int RO int TSCM had a significant positive correlation with the cell counts of all CCR5 gene edited cells, including TEM, which was not observed for the proliferation rates of other subsets (Fig. 6B). This suggests that self-renewal of CCR5 gene edited CD45RA int RO int TSCM is important for replenishment and maintenance of CCR5 mutations in other memory subsets.
• a CD45RA+ TSCM subset was previously described with characteristics of conventional memory T cells, enhanced self-renewal and the capacity to differentiate into other memory subsets.
• a CD45RA+CD45RO+CCR7+CD27+CD95+ Tsc M -like phenotype has been previously reported following in-vitro expansion of purified CD4+ and CD8+ naive T cells that were co-stimulated in the presence of cytokines such as IL-2, IL-7, IL-15, or IL- 21; however, the potential of these cells to persist in-vivo and whether a proportion of these cells could revert to the CD45RA+CD45RO- phenotype was not investigated.
• CD45RA int RO int TSCM subset expressing low levels of CD45RA and CD45RO was seen to emerge in-vivo upon ART initiation combined with IL-2 therapy that correlated with CD4+ T cell increases, demonstrating that CD45RA int RO int cells can also be generated in-vivo in response to homeostatic proliferation.
• the CD45RA int RO int TSCM subset we have identified in this study, also demonstrated the capacity of self-renewal as observed by geneset enrichment of the Wnt-signaling cascade.
• TSCM have previously been shown to be permissive to HIV-infection.
• the importance of limiting HIV infection in early memory cells for the preservation of CD4+ T cell homeostasis has been shown in non-human primates as well as in viremic non-progressor HIV-infected individuals.
• the presence of TSCM cells (enriched in CCR5 gene edited alleles (up to -40% in the periphery at years 3-4)) within secondary lymphoid tissues - where HIV replication would be partially if not totally inhibited- may lead to their long term survival .
• results from both SB-728-0902 and SB-728- 1101 studies confirm the role of SB-728-T infusion in restoration of T cell homeostasis and cognate help to HIV specific CD8 T cells that can lead to the differentiation of TSCM into TEM that are protected from HIV infection.
• the cognate help provided is further highlighted by the observed decay in HIV reservoir that negatively correlated with both the expansion of CD45RA int RO int TSCM cells and GAG- specific CD 8+ TTM IL-2 producing cells.
• the role of IL-2 production by HIV-specific CD8+ T cells on viral replication has previously been shown.

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