Vector packaging cell line
The invention relates to a method of increasing vector transduction in target cells. The invention provides for the recombinant engineering of a packaging cell line to be capable of expressing one or more membrane proteins which facilitate binding to, and activation of, a target cell. The invention also provides for recombinant engineering of a cell that endogenously expresses one or more such membrane proteins into a packaging cell line. A vector packaged into viral particles via use of such cell lines would comprise an outer envelope containing these proteins. The particles would be specifically suited for binding and targeting to a target cell to facilitate transduction thereof with the vector. The target cell may also be simultaneously activated (stimulated) by the packaged vector in the absence of exogenously supplied stimulatory molecules.
1 . A recombinant retroviral packaging cell comprising a first nucleic acid molecule capable of expressing, in said packaging cell, at least one membrane associated non-viral ligand which binds a cell surface molecule of a target cell,
wherein said cell produces no viral particles in the absence of a second nucleic acid molecule.
2 . The cell according to claim 1 wherein said retroviral packaging cell comprises
i) a heterologous nucleic acid molecule capable of expressing a viral ligand which binds a cell surface molecule of a target cell and which optionally functions to mediate fusion of a cell membrane containing said viral ligand with said target cell; or
ii) a heterologous nucleic acid molecule which is capable of expressing CD49d, CD54, CD80, CD86, or a combination thereof.
3 . The cell according to claim 1 wherein said target cell is an antigen presenting cell (APC), a cell of the hematopoietic lineage, a stem cell, a hematopoietic stem cell, a lymphocyte, a neuron, an endothelial cell, or a tumor cell.
4 . The cell according to claim 1 wherein said at least one membrane associated non-viral ligand(s) is
i) a co-stimulatory molecule that binds a T cell surface molecule to activate T cell proliferation when the CD3/TCR complex of said T cell is bound by a natural or artificial ligand or by a specific Ab, and/or
ii) a molecule that plays a role in cell-cell adhesion via binding to said cell surface molecule.
5 . The cell according to claim 1 wherein
i) said at least one membrane associated non-viral ligand(s) activates said target cell to proliferate after binding said cell surface molecule;
ii) said at least one membrane associated non-viral ligand(s) comprise two such ligands;
iii) said membrane associated non-viral ligand(s) is that of a hematopoietic cell;
iv) said membrane associated non-viral ligand(s) are transmembrane protein(s); or
v) said membrane associated non-viral ligand(s) is CD28 or CD28BP or CD40 or CD62L or CD80 (B7-1) or CD86 (B7-2) or Fas ligand (FasL) or CD70 or LFA-3 (CD58) or B7-H1 (PD-L1) or B7-H2 or B7-H3 (B7RP-2) or B7-H4 or CD2 or CD3 or CD3/TCR complex or CD11a or CD26 or CD27 or CD28 or CD30L or CD32 or CD38 or CD40L (CD154) or CD45 or CD49 or CD50 (ICAM-3) or CD54 (ICAM-1) or CD100 or CD122 or CD137L (4-1BB Ligand) or CD153 or CTLA-4 (CD152) or ICOS or OX40L (CD134) or PD-1 or PD-L2 (B7-DC) or SLAM (CD150) or TIM-1 or TIM-2 or TIM-3 or TIM-4 or 2B4 (CD244), or a combination thereof.
6 . The cell according to claim 1 wherein
i) said membrane associated non-viral ligand(s) is a B7 related molecule such as B7-H 1 , B7-H 2 (also known as ICOS-L, B7RP-1, and GL50), B7-H 3 , and B7-H 4 ;
ii) said membrane associated non-viral ligand(s) binds LFA-1, or a complex of CD11a with CD18, such as ICAM-2 (CD102), ICAM-3 (CD50), ICAM-4 (LW), or ICAM-5 (telencephalin); or
iii) said membrane associated non-viral ligand(s) is a PD-1 ligand, such as PD-L 2 or PD-L 1 ; an OX40 ligand (CD154) which binds OX40 (CD134); a 4-1BB ligand which binds 4-1BB (CD137); a ligand which binds LFA-2 (CD2), such as CD15, CD48, CD58, or CD59; a ligand which binds CD5, such as CD72; or a ligand which binds LFA-3 (CD58), such as CD2;
iv) said membrane associated non-viral ligand(s) is an antibody or antibody fragment that binds LFA-1, CD18, CD11b, CD11c, CD11d or CD43; or
v) said membrane associated non-viral ligand(s) is a microbial protein that binds LFA-1, CD18, CD11b, CD11c, CD11d or CD43.
7 . The cell according to claim 2 , wherein said retroviral packaging cell comprises a heterologous nucleic acid molecule which is capable of expressing the polio virus receptor CD155, the wild type herpes simplex virus (HSV)-1 envelope protein, or another viral envelope protein; and
i) the target cell is a hematopoietic stem cell;
ii) the target cell is CD34+, CD33+, CD14+, or expresses nectin 1;
iii) the target cell is a T cell;
iv) the target cell is a dendritic cell or a B cell in the germinal center of a lymph node;
v) the target cell is a fibroblast.
8 . A method of producing a recombinant retroviral packaging cell according to claim 1 , said method comprising
introducing, into a cell, a nucleic acid molecule capable of expressing, in said packaging cell, a viral gene product necessary for packaging and/or replication of a retrovirus, and
at least one nucleic acid molecule capable of expressing, in said packaging cell, at least one membrane associated non-viral ligand(s) which bind a cell surface molecule of a target cell.
9 . A method of packaging a viral vector, said method comprising
introducing a second nucleic acid molecule encoding or comprising a retroviral vector into a recombinant cell according to claim 1 , and
culturing said cell under conditions wherein said cell packages said vector into a particle comprising said membrane associated non-viral ligand(s) which bind a cell surface molecule of a target cell.
10 . A retroviral vector packaging cell comprising a cell that endogenously expresses at least one membrane associated ligand capable of binding to a cell surface molecule of a target cell or tissue.
11 . The packaging cell of claim 10 wherein the cell interacts with the target cell or tissue in vivo.
12 . The packaging cell of claim 11 wherein the cell is a bone marrow stromal cell and the target cell is a hematopoietic stem cell.
13 . The packaging cell of claim 10 wherein the ligand is an integrin and the target cells are CD34+ cells.
14 . The packaging cell of claim 13 wherein the ligand is SDF-1, VLA-4, VLA-5, or LFA-1, and the hematopoietic stem cell is CD34+ and CD38−/CXCR4+, or CD34+ and CD38 low/CXCR4+.
15 . The cell according to claim 10 wherein said retroviral packaging cell comprises a heterologous nucleic acid molecule capable of expressing a viral ligand which binds a cell surface molecule of a target cell and which optionally functions to mediate fusion of a cell membrane containing said viral ligand with said target cell.
16 . The cell according to claim 1 wherein said cell is a T cell or a cell of a T cell line.
17 . The cell according to claim 1 wherein said cell expresses CD86 but not CD54.
18 . A recombinant retroviral packaging cell comprising a first nucleic acid molecule capable of expressing, in said packaging cell, at least one membrane associated non-viral ligand which binds a cell surface molecule of a target cell and activates or stimulates the cell into cell cycle transition.
19 . The cell according to claim 18 wherein said packaging cell expresses a retroviral structural protein or accessory protein.
20 . The cell according to claim 18 further comprising a retroviral vector to be packaged.