METHODS OF MANUFACTURING GENETICALLY-MODIFIED LYMPHOCYTES
The present disclosure relates generally to immunization and immunotherapy for the treatment or prevention of HIV. In particular, the methods include purifying peripheral blood mononuclear cells (PBMC) from a source, stimulating the PBMC with at least one HIV-specific peptide, depleting at least one subset of cells from the PBMC, wherein the at least one subset of cells comprises any one or more of CD8+ T cells, CD4+ T cells, γδ cells, NK cells, B cells, T regulatory cells, and NKT cells, transducing the depleted PBMC with a viral delivery system encoding at least one genetic element, culturing the transduced PBMC for at least one day, and harvesting the cultured PBMC.
1 . A method comprising:
purifying peripheral blood mononuclear cells (PBMC) from a source;
stimulating the PBMC with at least one HIV-specific peptide;
depleting at least one subset of cells from the PBMC, wherein the at least one subset of cells comprises any one or more of CD8+ T cells, γδ cells, NK cells, B cells, T regulatory cells, and NKT cells;
transducing the depleted PBMC with a viral delivery system encoding at least one genetic element;
culturing the transduced PBMC for at least one day; and
harvesting the cultured PBMC.
2 . The method of claim 1 , wherein the culturing occurs in a static culture system or a semi-static culture system.
3 . The method of claim 1 , wherein the at least one HIV-specific peptide comprises a pool of synthetic, overlapping peptides.
4 . The method of claim 3 , wherein the pool of synthetic, overlapping peptides represents the HIV Gag polyprotein.
5 . The method of claim 1 , wherein the depleting comprises separating the at least one subset of cells from the depleted PBMC.
6 . The method of claim 5 , wherein the separating comprises magnetic bead separation.
7 . The method of claim 1 , wherein the at least one genetic element comprises a small RNA capable of inhibiting production of chemokine receptor CCR5 or at least one small RNA capable of targeting an HIV RNA sequence.
8 . The method of claim 7 , wherein the HIV RNA sequence comprises an HIV Vif sequence, an HIV Tat sequence, or a variant thereof.
9 . The method of claim 7 , wherein the at least one genetic element comprises a microRNA or a shRNA.
10 . The method of claim 7 , wherein the at least one genetic element comprises a microRNA having at least 80%, or at least 85%, or at least 90%, or at least 95% identity with at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 30.
11 . A genetically-modified lymphocyte for treatment of a subject infected with HIV prepared by a process comprising the steps of:
purifying peripheral blood mononuclear cells (PBMC) from the subject, wherein the PBMC comprise at least one lymphocyte;
stimulating the at least one lymphocyte with at least one HIV-specific peptide;
depleting at least one subset of cells from the PBMC, wherein the at least one subset of cells comprises any one or more of CD8+ T cells, CD4+ T cells, γδ cells, NK cells, B cells, T regulatory cells, and NKT cells;
transducing the at least one lymphocyte with a viral delivery system encoding at least one genetic element;
culturing the PBMC for at least one day; and
harvesting the cultured PBMC.
12 . A genetically-modified lymphocyte for treatment of a subject immunized with a preventive or therapeutic HIV vaccine prepared by a process comprising the steps of:
purifying peripheral blood mononuclear cells (PBMC) from the subject, wherein the PBMC comprise at least one lymphocyte;
stimulating the at least one lymphocyte with at least one HIV-specific peptide;
depleting at least one subset of cells from the PBMC, wherein the at least one subset of cells comprises any one or more of CD8+ T cells, CD4+ T cells, γδ cells, NK cells, B cells, T regulatory cells, and NKT cells;
transducing the at least one lymphocyte with a viral delivery system encoding at least one genetic element;
culturing the PBMC for at least one day; and
harvesting the cultured PBMC.
13 . A genetically-modified lymphocyte for treatment of a subject exposed to, but not infected with, HIV prepared by a process comprising the steps of:
purifying peripheral blood mononuclear cells (PBMC) from the subject, wherein the PBMC comprise at least one lymphocyte;
stimulating the PBMC with at least one HIV-specific peptide;
depleting at least one subset of cells from the PBMC, wherein the at least one subset of cells comprises any one or more of CD8+ T cells, CD4+ T cells, γδ cells, NK cells, B cells, T regulatory cells, and NKT cells;
transducing the at least one lymphocyte with a viral delivery system encoding at least one genetic element;
culturing the PBMC for at least one day; and
harvesting the cultured PBMC.
14 . A method, comprising:
stimulating PBMC with at least one HIV-specific peptide;
depleting at least one subset of cells from the PBMC, wherein the at least one subset of cells comprises any one or more of CD8+ T cells, γδ cells, NK cells, B cells, T regulatory cells, and NKT cells; and
transducing the depleted PBMC with a viral delivery system encoding at least one genetic element.
15 . The method of claim 14 , wherein the at least one HIV-specific peptide comprises an HIV gag peptide.
16 . The method of claim 14 , wherein the at least one genetic element comprises a small RNA capable of inhibiting production of chemokine receptor CCR5 or at least one small RNA capable of targeting an HIV RNA sequence.
17 . The method of claim 16 , wherein the HIV RNA sequences comprises an HIV Vif sequence, an HIV Tat sequence, or a variant thereof.