Methods for inducing partial apoptosis using caspase polypeptides

Abstract

The technology relates in part to methods for inducing partial apoptosis of cells that express an inducible caspase polypeptide. The technology further relates in part to methods for inducing partial apoptosis of cells that express an inducible modi- fied caspase polypeptide, having a modified dose response curve to the multimeric ligand inducer. The technology also relates in part to methods for cell therapy using cells that express the inducible caspase polypeptide or the inducible modified caspase poly- peptide, where the proportion of caspase polypeptide-expressing cells eliminated by apoptosis is related to the administered amount of the multimeric ligand inducer.

Description

DEMANDE OU BREVET VOLUMINEUX LA PRÉSENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND PLUS D'UN TOME. CECI EST LE TOME 1 DE 2 CONTENANT LES PAGES 1 À 206 NOTE: Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS / PATENTS THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE THAN ONE VOLUME THIS IS VOLUME 1 OF 2 CONTAINING PAGES 1 TO 206 NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME : NOTE POUR LE TOME / VOLUME NOTE: METHODS FOR INDUCING PARTIAL APOPTOSIS USING CASPASE POLYPEPTIDES Field The technology relates in part to methods for inducing partial apoptosis of cells that express an inducible caspase polypeptide. The technology further relates in part to methods for inducing partial apoptosis of cells that express an inducible modified caspase polypeptide, having a modified dose response curve to the multimeric ligand inducer. The technology also relates in part to methods for cell therapy using cells that express the inducible caspase polypeptide or the inducible modified caspase polypeptide, where the proportion of caspase polypeptide-expressing cells eliminated by apoptosis is related to the administered amount of the multimeric ligand inducer. Related Applications Priority is claimed to U.S. Provisional Patent Application serial number 61 / 831,428, filed June 5, 2013, and entitled "METHODS FOR INDUCING PARTIAL APOPTOSIS USING MODIFIED CASPASE POLYPEPTIDES," and to U.S. Provisional Patent Application serial number 61 / 949,847, filed March 7, 2014, and entitled 'METHODS FOR INDUCING PARTIAL APOPTOSIS USING MODIFIED CASPASE POLYPEPTIDES", This application is related to International Application Number PCT / US2014 / 022004 filed March 7, 2014, entitled MODIFIED CASPASE POLYPEPTIDES AND USES THEREOF. Background In most T cell therapies, such as therapies using chimeric antigen receptor-expressing T cells (CAR), donor lymphocyte infusions (DLIs), or T cell add-back following hematopoietic stem cell transplants (HSCTs), the clinical relevance of demonstrated efficacy against tumors is somewhat diminished by the risk of off-target or off-organ adverse effects. Moreover, overzealous on-target effects, such as those directed against large tumor masses, can lead to cytokine storms, associated with tumor lysis syndrome (TLS), cytokine release syndrome (CRS) or macrophage activation syndrome (MAS). As a result, there is great interest in the development of a stable, reliable "suicide gene" that can eliminate transferred T cells or stem cells in the event that they trigger serious adverse events (SAEs), or become obsolete following treatment. Methods for selectively killing therapeutic cells by inducing selective apoptosis, should an adverse event occur, are discussed in U.S. Patent Application Serial Number 13 / 112,739, filed May 20, 2011, and entitled METHODS FOR INDUCING SELECTIVE APOPTOSIS, naming Malcolm K. Brenner as inventor. Modified caspase-9 polypeptides are discussed in U.S. Patent Application Serial Number 13 / 792,135, filed March 10, 2013, and entitled MODIFIED CASPASE POLYPEPTIDES AND USES THEREOF, naming David Spencer, et al., as inventors. There is a need for a method for balancing the ability to rapidly remove the possible negative effects of donor cells used in cellular therapy, while retaining part or all of the beneficial effects of the therapy. Summary Upon an adverse event following cellular therapy, a discrete fraction of cells may be eliminated by partial apoptosis, allowing beneficial effects of the cellular therapy to remain. An example of a cellular therapy is adoptive T cell transfer after CD34<com:Sup>+< / com:Sup> stem cell transplantation. Administering T cells after stem cell transfer helps to accelerate the reconstitution of an immune system in the patient recipient. The T cells may be obtained from, for example, a matched related or unrelated donor. When a matched related or unrelated donor is not available, or the disease is too aggressive for an extensive donor search, the use of an HLA haploidentical family donor may be effective. Such donors may be parents, siblings, or second-degree relatives. Such infusions may enhance immune recovery and thereby reduce virus infections and eliminate relapsing leukemia cells. However, the coexistence of alloreactive T cells in a donor stem cell graft may cause graft- versus-host disease (GvHD) in which the donor cells react against the recipient, which may progressively damage the skin, gut, liver, and other organs of the recipient, often with fatal consequences. An inducible caspase-9 system may be applied to human T cells, which are then administered to stem cell transplantation patients. Upon exhibiting graft versus host disease symptoms, caspase-9 is activated after the administration of a multimeric ligand, which causes dimerization of the protein and induced apoptosis of the allogeneic activated T cells. A caspase-9 based apoptotic safety switch may also be applied to therapeutic chimeric antigen receptor-expressing cells, which express artificial receptors designed to convey antigen specificity to T cells. They include an antigen-specific component, a transmembrane component, and an intracellular component selected to activate the T cell and provide specific immunity. Chimeric antigen receptor-expressing T cells may be used in various therapies, including cancer therapies. While effective against tumors, in some cases these therapies have led to side effects due, in part to non-specific attacks on healthy tissue. An inducible caspase-9 system may be provided in these therapeutic cells before administering them to a patient, to provide the ability to selectively kill the therapeutic cells if the patient experiences negative side effects, such as, for example, on-target but off-organ toxicity, where the wrong organ is targeted by the chimeric antigen receptor. In other embodiments, caspase-9 based apoptotic safety switch may be used for the elimination of tissue stem cells and their progeny, and for augmentation of oncolytic virus-mediated tumor killing where too complete killing might limit the oncolytic effect. Methods featured in some embodiments include methods of inducing apoptosis in discrete fractions of caspase-9-expressing cells. By using these methods, for example, upon the occurrence of graft vs. host disease, a percentage of therapeutic cells causing the graft vs. host disease may be eliminated, while leaving a sufficient number of therapeutic cells to assist in the reconstitution of the patient's immune system. In another example, upon off target toxicity following transplantation, a percentage of chimeric antigen receptor-expressing therapeutic cells may be eliminated, while leaving a sufficient number of the cells to continue their therapeutic effect. In yet another example, where both therapeutic cells, such as chimeric antigen-receptor expressing cells and therapeutic T cells following stem cell transplantation are transfused in the patient, one of the populations of therapeutic cells may be eliminated upon the occurrence of an adverse event, without significantly affecting the proportion of the other population of therapeutic T cells. Thus, featured in some embodiments are methods for controlling the survival of transplanted therapeutic cells in a subject, comprising preparing or obtaining therapeutic cells; transfecting or transducing the therapeutic cells with a nucleic acid that encodes a chimeric protein comprising a multimeric ligand binding region and a caspase-9 polypeptide or a modified caspase-9 polypeptide, wherein the caspase-9 polypeptide or the modified caspase-9 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9; transplanting the transduced or transfected therapeutic cells into the subject; and after transplantation, administering an effective amount of a multimeric ligand that binds to the multimeric ligand binding region to the subject, wherein less than 80% of transplanted therapeutic cells that express the caspase-9 polypeptide or the modified caspase-9 polypeptide are killed following administration of the multimeric ligand; wherein the modified caspase-9 polypeptide has a reduced IC<com:Sub>50< / com:Sub> and / or an elongated dose response curve in response to the multimeric ligand, when compared to a caspase-9 polypeptide that is not modified. In some embodiments, the therapeutic cells are selected from the group consisting of hematopoietic stem cells, inducible progenitor cells (iPS), embryonic stem (ES) cells, mesenchymal stem cells, plasma (B) cells, myocytes, tumor infiltrating lymphocytes, and T cells. In some embodiments, the subject has undergone a stem cell transplant, for example, a transplant that is haplo-identical, matched unrelated, or matched related. In certain embodiments, the subject has been diagnosed with a hyperproliferative disease. In other embodiments, the subject has been diagnosed with an immune disease. In some embodiments, the modified caspase-9 polypeptide comprises an amino acid substitution selected from the group consisting of the caspase variants in Table 3. In some embodiments, the modified caspase-9 polypeptide comprises an amino acid substitution selected from the group consisting of N405Q, F404Y, F406L, F406T, F404W, and the caspase-9 polypeptide containing the substitution of dimerization domain amino acid residues 402-406 (GCFNF)-from caspase-9 with the equivalent position residues of Caspase10(GCFNF<com:Sup>402< / com:Sup>ISAQT). In some embodiments, the modified caspase-9 polypeptide comprises an amino acid substitution selected from the group consisting of D330A and T317A. In some embodiments, the modified caspase-9 polypeptide comprises an amino acid substitution selected from the group consisting of T317S, S144A, S144D, S196A, S183A, and S195A, the modified caspase-9 polypeptide comprises an amino acid substitution selected from the group consisting of D330A-N405Q, D330A-S144A, D330A-S144D, D330A- S196A, D330A-T317A, and D330A-S183A, the modified caspase-9 polypeptide comprises an amino acid substitution selected from the group consisting of F404T, F404W, N405F, and F406T, the modified caspase-9 polypeptide comprises an amino acid substitution selected from the group consisting of D315A, A316G, T317S, F319W, and S307A, the modified caspase-9 polypeptide comprises an amino acid substitution selected from the group consisting of Y153A and Y153F, the modified caspase-9 polypeptide comprises an amino acid substitution selected from the group consisting of C403S, C403T, C403, N405A, N406A, N406Y, and F406W, the modified caspase-9 polypeptide comprises an amino acid substitution selected from the group consisting of T317A, T317C, F318A, F319A, and the caspase-9 polypeptide containing the substitution of amino acid residues from caspase-9 with the equivalent position residues of Caspase10(402 ISAQT), and the caspase-9 polypeptide containing the substitution of amino acid residues from caspase-9 with the equivalent position Smac / DIABLO (ATPF<com:Sup>316< / com:Sup>AVPI), the modified caspase-9 polypeptide comprises an amino acid substitution selected from the group consisting of N405T, S317E, and D330A- N405T, the modified caspase-9 polypeptide comprises an amino acid substitution selected from the group consisting of F319W, F404Y, A316G, Y153A, F406L, C403A, N405Q, F406T, and the caspase-9 polypeptide containing the substitution of dimerization domain amino acid residues 402- 406 (GCFNF)-from Caspase with the equivalent position residues of Caspase 10 (ISAQT), or the modified caspase-9 polypeptide comprises an amino acid substitution selected from the group consisting of N405Q, ,F404W, F404Y, and F406T. Also provided are methods for controlling the survival of transplanted therapeutic cells in a subject, comprising preparing or obtaining therapeutic cells; transfecting or transducing a first subset of the therapeutic cells with a nucleic acid that encodes a chimeric protein comprising a multimeric ligand binding region and a first caspase-9 polypeptide, wherein the first caspase-9 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9; transfecting or transducing a second subset of the therapeutic cells with a nucleic acid that encodes a chimeric protein comprising a multimeric ligand binding region and a second caspase-9 polypeptide, wherein the second caspase-9 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9; transplanting the transduced or transfected first and second therapeutic cells into the subject; and after transplantation, administering an effective amount of a multimeric ligand that binds to the multimeric ligand binding region to the subject, wherein more of the therapeutic cells that express the first caspase-9 polypeptide are killed than the therapeutic cells that express the second caspase-9 polypeptide, following administration of the multimeric ligand. In some embodiments, the first caspase-9 polypeptide has a reduced IC<com:Sub>50< / com:Sub> and an elongated dose response curve in response to the multimeric ligand, when compared to the second caspase-9 polypeptide. In some embodiments, wherein the therapeutic cells are selected from the group consisting of hematopoietic stem cells, inducible progenitor cells (iPS), embryonic stem (ES) cells, mesenchymal stem cells, plasma (B) cells, myocytes and T cells. In some embodiments, the subject has been diagnosed with a hyperproliferative disease. In some embodiments, the subject has been diagnosed with an immune disease. In some embodiments, the first or second caspase- 9 polypeptide comprises an amino acid substitution selected from the group consisting of F319W, F404Y, A316G, Y153A, F406L, C403A, N405Q, C285A, F406T, or the caspase-9 polypeptide comprises ISAQT, the corresponding amino acid sequence of the dimerization domain in Caspase 10, or the first or second caspase-9 polypeptide comprises an amino acid substitution selected from the group consisting of N405Q, C285A, and F406T. In some embodiments, the second caspase-9 polypeptide has an IC<com:Sub>50< / com:Sub> for the multimeric ligand greater than 0.01 pM, 0.05 pM, 0.1 pM, 0.5 pM, 0.01 nM, 0.05 nM, 0.1nM, 0.5 nM, or 1 nM. In some embodiments, a therapeutically effective level of the first therapeutic cells comprising the chimeric antigen receptor remains active in the subject following administration of the multimeric ligand. In some embodiments, the second therapeutic cells are T cells, for example, T cells administered to a subject following stem cell transplantation. In some embodiments, the T cells are allodepleted before administration to the subject. In some embodiments, the T cells are not allodepleted before administration to the subject. In some embodiments, the second therapeutic cell comprises a chimeric antigen receptor. In some embodiments, the first therapeutic cells are T cells. In some embodiments, the second therapeutic cells are T cells administered to a subject following stem cell transplantation. In some embodiments, the T cells are allodepleted before administration to the subject. In other embodiments, the T cells are not allodepleted before administration to the subject. In certain embodiments, less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of transplanted therapeutic cells that express the caspase-9 polypeptide or the modified caspase-9 polypeptide are killed following administration of the multimeric ligand. In some embodiments, the modified caspase-9 polypeptide has an IC<com:Sub>50< / com:Sub> for the multimeric ligand greater than 0.01 pM, 0.05 pM, 0.1 pM, 0.5 pM, 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, or 1 nM. In some embodiments, the multimeric ligand binding region is selected from the group consisting of FKBP ligand-binding region, cyclophilin receptor ligand-binding region, steroid receptor ligand-binding region, cyclophilin receptor ligand-binding region, and tetracycline receptor ligand-binding region. In some embodiments, the ligand-binding region comprises a <com:Math><semantics>FvFvls<annotation encoding="application / x-tex">F_v F_{vls}< / annotation>< / semantics>< / com:Math> amino acid sequence. In some embodiments, the ligand is a small molecule. In some embodiments, the ligand is dimeric. In some embodiments, the ligand is dimeric FK506, or a dimeric FK506-like analog. In certain embodiments, the multimeric ligand is AP1903. In some embodiments, the multimeric ligand is AP20187. In some embodiments, the cells are T cells. In some embodiments, the chimeric protein further comprises a marker polypeptide. In some embodiments, the methods further comprise a selection step, wherein cells that express the marker are selected for administration to the subject. In some embodiments, the methods further comprise administering a second dose of the multimeric ligand to the subject, wherein the second dose comprises more multimeric ligand than the first dose. In some embodiments, multiple doses of multimeric ligand are administered to the subject, with an escalation of dosage levels among the multiple doses. In some embodiments, the escalation of dosage levels increases the number of therapeutic cells that are killed. In some embodiments, the dose is escalated from 0.01 to 1 mg / kg. In some embodiments, the doses are administered in increments of about 15 to 30 minutes. In some embodiments, the multimeric ligand is administered using a continuous infusion pump, and the concentration of multimeric ligand is increased during the infusion. In some embodiments, the concentration of multimeric ligand is increased until the desired percentage of therapeutic cells is killed. In some embodiments, the subject has graft vs. host disease and the administration of the multimeric ligand alleviates the disease. In some embodiments, the subject is human. In some embodiments, the therapeutic cell comprises a chimeric antigen receptor. In some embodiments, the subject exhibits symptoms of off-target toxicity before administration of the multimeric ligand. In other embodiments, the subject exhibits symptoms of tumor lysis syndrome (TLS), cytokine release syndrome (CRS) or macrophage activation syndrome (MAS) before administration of the multimeric ligand. In some embodiments, the administration of the multimeric ligand alleviates the off-target or off-organ toxicity. A discussion of off-target toxicity is provided in, for example, Heslop, H.E., Blood 122:853-854 (2013). In other embodiments, the administration of the multimeric ligand alleviates the tumor lysis syndrome (TLS), cytokine release syndrome (CRS) or macrophage activation syndrome (MAS). In some embodiments, a therapeutically effective level of therapeutic cells comprising the chimeric antigen receptor remains active in the subject following administration of the multimeric ligand. In some embodiments, the patient has cancer. In some embodiments, the patient has a solid tumor. In some embodiments, the cancer is present in the blood or bone marrow of the patient. In some embodiments, the patient has a blood or bone marrow disease. In some embodiments, the patient has been diagnosed with any condition or disorder that can be alleviated by stem cell transplantation. In some embodiments, the patient has been diagnosed with sickle cell anemia or metachromatic leukodystrophy. In some embodiments, the promoter is activated in activated T cells. In certain embodiments, the caspase-9 polypeptide is a truncated caspase-9 polypeptide, or the caspase-9 polypeptide lacks the Caspase recruitment domain. In some embodiments, the patient exhibits one or more Stage 1, 2, 3, or 4 graft versus host disease symptoms. In some embodiments, after administration of the multimeric ligand, the number of alloreactive T cells is reduced. In some embodiments, the alloreactive T cells express a marker and CD3. In some embodiments, the number of alloreactive T cells is reduced by about 90% or more after administration of the multimeric ligand. In some embodiments, after administration of the multimeric ligand, donor T cells survive in the patient that are able to expand and are reactive to viruses and fungi. In some embodiments, after administration of the multimeric ligand, donor T cells survive in the patient that are able to expand and are reactive to tumor cells in the patient. In some embodiments, the patients have received haplo-CD34<com:Sup>+< / com:Sup> stem cell transplants before or at the same time as administration of the donor T cells. In some embodiments, the inducible chimeric caspase-9 polypeptide has been modified to have a different sensitivity to the ligand inducer, or to have a different basal activity in the transduced or transfected cell, when compared to wild type caspase-9 polypeptide, or wild type caspase-9 polypeptide that has been truncated to remove the CARD domain. Thus in certain embodiments, the methods of the present application use chimeric polypeptides comprising modified caspase-9 polypeptides, including, for example, iCasp9 D330A, iCasp9 N405Q, and iCasp9 D330A N405Q, demonstrated low to undetectable basal activity, respectively, with a minimum deleterious effect on their AP1903 IC<com:Sub>50< / com:Sub> in a SEAP reporter-based, surrogate killing assay. In some embodiments, a cell is provided which comprises a polynucleotide that encodes a chimeric protein comprising a multimerization region and a modified caspase-9 polypeptide, wherein the modified caspase-9 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9, and comprises at least one amino acid substitution selected from the group consisting of S144A, S144D, Y153A, Y153F, S183A, S195A, S196A, S196D, S307A, D315A, A316G, T317A, T317C, T317E, T317S, P318A, F319A, F319W, F326K, D327G, D327K, D327R, Q328K, Q328R, L329E, L329G, L329K, D330A, D330E, D330G, D330N, D330S, D330V, A331K, C403A, C403S, C403T, F404T, F404W, F404Y, N405A, N405F, N405Q, N405T, F406A, F406T, F406W, F406Y, G402A, G402I, G402Q, G402Y, C403P, F404A, F404S, and F406L. In some embodiments, the at least one amino acid substitution is selected from the group consisting of S144A, S144D, Y153A, Y153F, S183A, S195A, S196A, S196D, S307A, D315A, A316G, T317A, T317C, T317E, T317S, P318A, F319A, F319W, F326K, D327G, D327K, D327R, Q328K, Q328R, L329E, L329G, L329K, D330A, D330E, D330G, D330N, D330S, D330V, A331K, C403A, C403S, C403T, F404T, F404W, F404Y, N405A, N405F, N405Q, N405T, F406A, F406T, F406W, and F406Y. In some embodiments, the at least one amino acid substitution is selected from the group consisting of S144A, S144D, Y153A, Y153F, S183A, S195A, S196A, S196D, S307A, D315A, A316G, T317A, T317C, T317S, P318A, F319A, F319W, L329E, D330A, D330E, D330G, D330N, D330S, D330V, C403A, C403S, C403T, F404T, F404W, F404Y, N405A, N405F, N405Q, F406A, F406T, F406W, and F406Y. In some embodiments, the at least one amino acid substitution is S144A, S144D, Y153A, Y153F, S183A, S195A, S196A, S196D, S307A, D315A, A316G, T317A, T317S, F319W, L329E, D330A, D330E, D330G, D330N, D330S, D330V, F404T, F404W, F404Y, N405F, N405Q, F406A, F406T, F406W, and F406Y. In some embodiments, the at least one amino acid substitution is S144A, S144D, S183A, S195A, S196A, S196D, T317A, T317S, L329E, D330A, D330E, D330G, D330N, D330S, D330V, F404Y, N405Q, F406A, F406W, and F406Y. In some embodiments, the at least one amino acid substitution is selected from the group consisting of T317S, S144A, S133, and S196D. In some embodiments, the at least one amino acid substitution is selected from the group consisting of S183A, S195A, S196A, S196D, T317A, L329E, D330A, D330E, D330G, D330N, D330S, D330V, F404Y, N405Q, F406A, F406W, and F406Y. In some embodiments, the at least one amino acid substitution is D330A. In some embodiments, the at least one amino acid substitution is D330E. In some embodiments, the at least one amino acid substitution is N405Q. In some embodiments, the modified caspase-9 polypeptide comprises at least two amino acid substitutions selected from the group consisting of D330A-N405Q, D330A-S144A, D330A-S144D, D330A-S183A, D330A-S196A, N405Q-S144A, N405Q-S144D, N405Q-S196D, N405Q-T317S, N405Q-S144Aco, N405Q- T317Sco, 402GCFNF406ISAQT (CASP-10), 316ATPF319AVPI (SMAC / Diablo), D330A-N405T, D315A- D330A, D330A-Y153A, D330A-Y153F, D330A-T317E, 402GCFNF406CIVSM (CASP-3), <com:Sup>402< / com:Sup>GCFNF<com:Sup>406< / com:Sup>AAAAA, <com:Sup>402< / com:Sup>GCFNF<com:Sup>406< / com:Sup>YCSTL (CASP-2), and <com:Sup>402< / com:Sup>GFNF<com:Sup>406< / com:Sup>QPTFT (CASP-8). In some embodiments, the modified caspase-9 polypeptide comprises at least two amino acid substitutions selected from the group consisting of D330A-N405Q, D330A-S144A, D330A-S144D, D330A- S183A, D330A-S196A, N405Q-S144A, N405Q-S144D, N405Q-S196D, N405Q-T317S, N405Q- S144Aco, N405Q-T317Sco, 402GCFNF406ISAQT (CASP-10), 316ATPF319AVPI (SMAC / Diablo), and D330A-N405T. In some embodiments, the modified caspase-9 polypeptide comprises at least two amino acid substitutions selected from the group consisting of D330A-N405Q, D330A-S144A, D330A-S144D, D330A-S183A, D330A-S196A, N405Q-S144A, N405Q-S144D, N405Q-S196D, N405Q-T317S, N405Q-S144Aco, N405Q-T317Sco, 402GCFNF406ISAQT (CASP-10), and <com:Sup>316< / com:Sup>ATPF<com:Sup>319< / com:Sup>AVPI (SMAC / Diablo). In some embodiments, the modified caspase-9 polypeptide comprises at least two amino acid substitutions selected from the group consisting of D330A- N405Q, D330A-S144A, D330A-S144D, D330A-S183A, D330A-S196A, N405Q-S144A, N405Q- S144D, N405Q-S196D, N405Q-T317S, N405Q-S144Aco, N405Q-T317Sco, and <com:Sup>402< / com:Sup>GCFNF<com:Sup>406< / com:Sup>ISAQT (CASP-10). In some embodiments, the modified caspase-9 polypeptide comprises at least two amino acid substitutions selected from the group consisting of N405Q- S144Aco and N405Q-T317Sco. In some embodiments, the at least one amino acid substitution is selected from the group consisting of S144A, S144D, Y153A, Y153F, S183A, S195A, S196A, S307A, D315A, A316G, T317A, T317C, T317E, T317S, P318A, F319A, F319W, F326K, D327G, D327K, D327R, Q328K, Q328R, L329E, L329G, L329K, D330A, D330E, D330G, D330N, D330S, D330V, A331K, F404T, F404W, F404YN405F, N405Q, and F406T. In some embodiments, the at least one amino acid substitution is selected from the group consisting of S144A, S144D, Y153A, Y153F, S183A, S195A, S196A, S307A, D315A, A316G, T317A, T317C, T317E, T317S, P318A, F319A, F319W, F326K, D327G, D327K, D327R, Q328K, Q328R, L329E, L329G, L329K, D330A, D330E, D330G, D330N, D330S, D330V, A331K, F404T, F404W, F404Y, N405F, N405Q, and F406T. In some embodiments, the at least one amino acid substitution is selected from the group consisting of S144A, S144D, Y153A, Y153F, S183A, S195A, S196A, S307A, D315A, A316G, T317A, T317C, T317S, P318A, F319A, F319W, L329E, D330A, D330E, D330G, D330N, D330S, D330V, F404T, F404W, F404Y, N405F, N405Q, and F406T. In some embodiments, the at least one amino acid substitution is selected from the group consisting of S144A, S144D, Y153A, Y153F, S183A, S195A, S196A, S307A, D315A, A316G, T317A, T317S, F319W, D330A, F404T, F404W, F404Y, N405F, N405Q, and F406T. In some embodiments, the at least one amino acid substitution is selected from the group consisting of S144A, S144D, S183A, S195A, S196A, T317A, T317S, D330A, F404Y, and N405Q. In some embodiments, the at least one amino acid substitution is selected from the group consisting of S196D, T317C, T317E, P318A, F319A, F326K, D327G, D327K, D327R, Q328K, Q328R, L329E, L329G, L329K, D330E, D330G, D330N, D330S, D330V, A331K, C403A, C403S, C403T, N405A, N405T, F406A, F406W, F406Y, G402A, G402I, G402Q, G402Y, C403P, F404A, F404S, and F406L. In some embodiments, the at least one amino acid substitution is selected from the group consisting of S196D, T317C, T317E, P318A, F319A, F326K, D327G, D327K, D327R, Q328K, Q328R, L329E, L329G, L329K, D330E, D330G, D330N, D330S, D330V, A331K, C403A, C403S, C403T, N405A, N405T, F406A, F406W, and F406Y. In some embodiments, the at least one amino acid substitution is selected from the group consisting of S196D, T317C, P318A, F319A, L329E, D330E, D330G, D330N, D330S, D330V, C403A, C403S, C403T, N405A, F406A, F406T, F406W, and F406Y. In some embodiments, the at least one amino acid substitution is selected from the group consisting of S196D, L329E, D330E, D330G, D330N, D330S, D330V, F406A, F406T, F406W, and F406Y. In some embodiments, the at least one amino acid substitution is selected from the group consisting of S196D, L329E, D330E, D330G, D330N, D330S, D330V, F406A, F406W, and F406Y. 47. The cell of claim 1, wherein, the at least one amino acid substitution is selected from the group consisting of N405Q, F404Y, F406A, F406W, F406Y, F404T, F404W, N405F, F406T, C403A, C403S, C403T, N405A, and N405T. In some embodiments, the at least one amino acid substitution is selected from the group consisting of N405Q, F404Y, F406A, F406W, and F406Y. In some embodiments, the at least one amino acid substitution is selected from the group consisting of T317S, D330A, D330E, D330G, D330N, D330S, D330V, L329E, T317A, D315A, A316G, T317C, P318A, F319A, T317E, F326K, D327G, D327K, D327R, Q328K, Q328R, L329G, L329K, and A331K. In some embodiments, the at least one amino acid substitution is selected from the group consisting of T317S, D330A, D330E, D330G, D330N, D330S, D330V, L329E, and T317A. In some embodiments, the at least one amino acid substitution is selected from the group consisting of S144A, S144D, S196D, S183A, S195A, S196A, Y153A, Y153F, and S307A. In some embodiments, the polynucleotide comprises optimized codons encoding the caspase-9 polypeptide, in some embodiments, the polynucleotide coding for the modified caspase-9 polypeptide comprises an amino acid substitution of N405Q, and comprises optimized codons. In some embodiments, the polynucleotide coding for the modified caspase-9 polypeptide comprises the nucleic acid sequence of SEQ ID NO: 39. Also provided are modified caspase polypeptides comprising the amino acid sequences discussed herein as part of the cells which comprise polynucleotides coding for the chimeric modified caspase-9 polypeptides. Also provided are nucleic acids comprising polynucleotides coding for the modified caspase-9polypeptides and the chimeric modified caspase-9 polypeptides. Also provided are vectors comprising the polynucleotides coding for the modified caspase polypeptides and the chimeric modified caspase polypeptides. In some embodiments, the cell is a human cell. The cell of the present application may be any type of eukaryotic cell, for example a mammalian cell, for example a horse, dog, cat, cow, or human cell. In some embodiments, the cell is a progenitor cell. In some embodiments, the cell is a hematopoietic progenitor cell. In some embodiments, the cell is selected from the group consisting of mesenchymal stromal cells, embryonic stem cells, and inducible pluripotent stem cells. In some embodiments, the cell is a T cell. In some embodiments, the cell is obtained or prepared from bone marrow. In some embodiments, the cell is obtained or prepared from umbilical cord blood. In some embodiments, the cell is obtained or prepared from peripheral blood. In some embodiments, the cell is obtained or prepared from peripheral blood mononuclear cells. In some aspects, the polynucleotide coding for the chimeric polypeptide or modified caspase-9 polypeptide is operably linked to a promoter. In some embodiments, the promoter is developmentally regulated and the caspase-9 polypeptide is expressed in developmentally differentiated cells. In some embodiments, the promoter is tissue-specific and the caspase-9 polypeptide is expressed in the specific tissue. In some embodiments, the promoter is activated in activated T cells. In some embodiments, the promoter comprises a 5'LTR sequence. In some embodiments, the chimeric protein further comprises a marker polypeptide, for example, but not limited to, a ΔCD19 polypeptide. In some embodiments, the caspase-9 polypeptide is a truncated caspase-9 polypeptide. In some embodiments, the caspase-9 polypeptide lacks the Caspase recruitment domain. In some embodiments, wherein the multimerization region is selected from the group consisting of FKBP, cyclophilin receptor, steroid receptor, tetracycline receptor, heavy chain antibody subunit, light chain antibody subunit, single chain antibodies comprised of heavy and light chain variable regions in tandem separated by a flexible linker domain, and mutated sequences thereof. In some embodiments, the multimerization region is an FKBP12 region. In some embodiments, the FKB12 region is an FKB12v<com:Sub>36< / com:Sub> region. In some embodiments, the multimerization region is Fv'FvIs. In some embodiments, the multimerization region binds a ligand selected from the group consisting of an FK506 dimer and a dimeric FK506 analog ligand. In some embodiments, the ligand is AP1903, in other embodiments, the ligand is AP20187. In some embodiments, wherein the multimerization region has an amino acid sequence of SEQ ID NO: 29 or a functional fragment thereof. In some embodiments, the multimerization region is encoded by a nucleotide sequence in SEQ ID NO: 30, or a functional fragment thereof. In some embodiments, the multimerization region further comprises a polypeptide having an amino acid sequence of SEQ ID NO: 32, or a functional fragment thereof. In some embodiments, the multimerization region further comprises a polypeptide encoded by a nucleotide sequence in SEQ ID NO: 31, or a functional fragment thereof. In some embodiments, the multimerization region further comprises a polypeptide having an amino acid sequence of SEQ ID NO: 32, or a functional fragment thereof. In some embodiments, the multimerization region further comprises a polypeptide encoded by a nucleotide sequence in SEQ ID NO: 31, or a functional fragment thereof. In some embodiments, the multimerization region further comprises a polypeptide having an amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 32, or a functional fragment thereof. In some embodiments, the multimerization region further comprises a polypeptide encoded by a nucleotide sequence in SEQ ID NO: 30 or SEQ ID NO: 31, or a functional fragment thereof. In some aspects of the present application, the cells are transduced or transfected with a viral vector. The viral vector may be, for example, but not limited to, a retroviral vector, such as, for example, but not limited to, a murine leukemia virus vector; an SFG vector; and adenoviral vector, or a lentiviral vector. In some aspects, the cells are further transfected or transduced with a gene expression vector. In some embodiments, the cells further comprise a polynucleotide that encodes the modified caspase-9 polypeptide and further comprise a second polynucleotide that encodes a heterologous polypeptide. In some embodiments, the heterologous polypeptide is a chimeric antigen receptor. In some embodiments, the cells further comprises a polynucleotide that encodes a chimeric protein comprising a multimerization region and a caspase-9 polypeptide or a second modified caspase-9 polypeptide., wherein the first and second caspase-9 polypeptides comprise different amino acid sequences and have different basal activities or different <com:Math><semantics>IC50<annotation encoding="application / x-tex">IC_{50}< / annotation>< / semantics>< / com:Math>s. In some aspects is provided a cell, wherein the modified caspase-9 polypeptide and the caspase-9 polypeptide; or the modified caspase-9 polypeptide and the second modified caspase-9 polypeptide have different IC<com:Sub>50< / com:Sub>s or different elongated dose response curves, or different <com:Math><semantics>C50<annotation encoding="application / x-tex">C_{50}< / annotation>< / semantics>< / com:Math>s and different elongated dose response curves to the multimeric ligand. In some embodiments, the cell is isolated. In some embodiments, the cell is in a human subject. In some embodiments, the cell is transplanted in a human subject. Also provided are methods of administering donor T cells to a human patient, comprising administering any of the cells of the present application to a human patient, wherein the cells are human donor T cells. In some embodiments, the cells are transduced or transfected in a donor cell culture. In some embodiments, the method further comprises detecting the presence of graft versus host disease in the patient after administering the cells to the patient; and administering a multimeric ligand that binds to the multimerization region to the patient for whom the presence of graft versus host disease is detected. In some embodiments, the effect of the graft versus host disease is reduced following administration of the multimeric ligand. Also provided are methods of stem cell transplantation, comprising administering a stem cell transplant to a human patient; and administering cells of the present application to the patient, wherein the cells are donor T cells, to the patient. In some embodiments, the stem cell transplant is selected from the group consisting of a matched transplant, a partially-matched transplant, a haploidentical transplant, and a CD34<com:Sup>+< / com:Sup> haploidentical stem cell transplant. In some embodiments, the human donor T cells are matched, partially matched, or haploidentical to the patient's T cells. Also provided in certain aspects are methods for controlling the survival of transplanted therapeutic cells in a patient, comprising administering cells of the present application to a human patient, and administering a multimeric ligand to the patient, wherein the multimeric ligand binds to the multimerization region, wherein the administered cells that express the caspase-9 polypeptide are killed following administration of the multimeric ligand. In some embodiments, the method comprises preparing cells of the present application for transplantation, and transplanting the therapeutic cells into the human patient. In some embodiments, the patient has cancer. In some embodiments, the patient has a solid tumor. In some embodiments, the cancer is present in the blood or bone marrow of the patient. In some embodiments, the patient has a blood or bone marrow disease. In some embodiments, the patient has been diagnosed with any condition or disorder that can be alleviated by stem cell transplantation. The methods of the present application may further comprise a selection step, wherein cells that express a marker are selected for administration to the patient. The marker may be, for example, but not limited to, <com:Math><semantics>Δ<annotation encoding="application / x-tex">\Delta< / annotation>< / semantics>< / com:Math>CD19. In some embodiments, the cells are transfected in a donor cell culture that is prepared from a bone marrow sample. In some embodiments, the cells are transfected in a donor cell culture that is prepared from peripheral blood. In some embodiments, the donor cell culture is prepared from donor peripheral blood mononuclear cells. In some embodiments, the donor T cells are allodepleted from the donor cell culture before transfection or transduction. In some embodiments, the donor T cells are not allodepleted from the donor cell culture before transfection or transduction. In some embodiments, the transduced or transfected T cells are cultured in the presence of IL-2 before administration to the patient. In certain embodiments, the methods of the present application further comprise administering a multimeric ligand that binds to the multimerization region, such as, for example, AP1903 or AP20187. In some embodiments, the multimeric ligand is administered to treat graft versus host disease. In some embodiments, the patient exhibits graft versus host disease symptoms before the multimeric ligand is administered. In some embodiments, the patient exhibits one or more Stage 0, Stage 1, Stage 2, Stage 3, or Stage 4 graft versus host disease symptoms. In certain embodiments of the methods, more than one dose of the multimeric ligand is administered. In some embodiments, after administration of the multimeric ligand, the number of alloreactive T cells is reduced. In some embodiments, the alloreactive T cells express the marker and CD3. In some embodiments, the number of alloreactive T cells is reduced by about 90% or more after administration of the multimeric ligand. In some embodiments, after administration of the multimeric ligand, donor T cells survive in the patient that are able to expand and are reactive to viruses and fungi. In some embodiments, after administration of the multimeric ligand, donor T cells survive in the patient that are able to expand and are reactive to tumor cells in the patient. In some embodiments, the patient has received stem cell transplants before or at the same time as administration of the donor T cells. In some embodiments, the stem cell transplant is haploidentical. In some embodiments, the donor T cells are haploidentical and are not allodepleted before administration to the patient. In some embodiments, at least 1 x 10<com:Sup>6< / com:Sup> transduced or transfected donor T cells are administered to the patient. In some embodiments, at least <com:Math><semantics>1×107<annotation encoding="application / x-tex">1 \times 10^7< / annotation>< / semantics>< / com:Math> transduced or transfected donor T cells are administered to the patient. In some embodiments, at least 1 x 108 transduced or transfected donor T cells are administered to the patient. In some embodiments, personalized treatment is provided wherein the stage or level of the disease or condition is determined before administration of the multimeric ligand, before the administration of an additional dose of the multimeric ligand, or in determining method and dosage involved in the administration of the multimeric ligand. These methods may be used in any of the methods of any of the diseases or conditions of the present application. Where these methods of assessing the patient before administering the ligand are discussed in the context of graft versus host disease, it is understood that these methods may be similarly applied to the treatment of other conditions and diseases. Thus, for example, in some embodiments of the present application, the method comprises administering therapeutic cells to a patient, and further comprises identifying a presence or absence of a condition in the patient that requires the removal of transfected or transduced therapeutic cells from the patient; and administering a multimeric ligand that binds to the multimerization region, maintaining a subsequent dosage of the multimeric ligand, or adjusting a subsequent dosage of the multimeric ligand to the patient based on the presence or absence of the condition identified in the patient. And, for example, in other embodiments of the present application, the method further comprises determining whether to administer an additional dose or additional doses of the multimeric ligand to the patient based upon the appearance of graft versus host disease symptoms in the patient. In some embodiments, the method further comprises identifying the presence, absence or stage of graft versus host disease in the patient, and administering a multimeric ligand that binds to the multimerization region, maintaining a subsequent dosage of the multimeric ligand, or adjusting a subsequent dosage of the multimeric ligand to the patient based on the presence, absence or stage of the graft versus host disease identified in the patient. In some embodiments, the method further comprises identifying the presence, absence or stage of graft versus host disease in the patient, and determining whether a multimeric ligand that binds to the multimerization region should be administered to the patient, or the dosage of the multimeric ligand subsequently administered to the patient is adjusted based on the presence, absence or stage of the graft versus host disease identified in the patient. In some embodiments, the method further comprises receiving information comprising the presence, absence or stage of graft versus host disease in the patient; and administering a multimeric ligand that binds to the multimerization region, maintaining a subsequent dosage of the multimeric ligand, or adjusting a subsequent dosage of the multimeric ligand to the patient based on the presence, absence or stage of the graft versus host disease identified in the patient. In some embodiments, the method further comprises identifying the presence, absence or stage of graft versus host disease in the patient, and transmitting the presence, absence or stage of the graft versus host disease to a decision maker who administers a multimeric ligand that binds to the multimerization region, maintains a subsequent dosage of the multimeric ligand, or adjusts a subsequent dosage of the multimeric ligand administered to the patient based on the presence, absence or stage of the graft versus host disease identified in the subject. In some embodiments, the method further comprises identifying the presence, absence or stage of graft versus host disease in the patient, and transmitting an indication to administer a multimeric ligand that binds to the multimeric binding region, maintain a subsequent dosage of the multimeric ligand or adjust a subsequent dosage of the multimeric ligand administered to the patient based on the presence, absence or stage of the graft versus host disease identified in the subject. In some aspects, methods are provided for treating graft versus host disease in a patient who has undergone cell therapy, wherein one or more of the cells introduced for the therapy is a cell of the present application, comprising administering a multimeric ligand that binds to the multimerization region to the patient. In some embodiments, after administration of the multimeric ligand that binds to the multimeric binding region, the number of alloreactive T cells is reduced. In some embodiments, alloreactive T cells that are not undergoing cell division are ablated. In some embodiments, within 2 hours of administration of the multimeric ligand, at least 90% of CD3<com:Sup>+< / com:Sup>ΔCD19<com:Sup>+< / com:Sup> cells are ablated. In some embodiments, within 1 hour of administration of the multimeric ligand, at least 90% of CD3<com:Sup>+< / com:Sup>ΔCD19<com:Sup>+< / com:Sup> cells are ablated. In some embodiments, within 30 minutes of administration of the multimeric ligand, at least 90% of CD3<com:Math><semantics>+Δ<annotation encoding="application / x-tex">^+\Delta< / annotation>< / semantics>< / com:Math>CD19<com:Math><semantics>+<annotation encoding="application / x-tex">^+< / annotation>< / semantics>< / com:Math> cells are ablated. In some embodiments, within 24 hours of administration of the multimeric ligand, there is a further log reduction of CD3<com:Sup>+< / com:Sup>ΔCD19<com:Sup>+< / com:Sup> cells compared to the amount of CD3<com:Sup>+< / com:Sup>ΔCD19<com:Sup>+< / com:Sup> cells at 30 minutes after administration of the multimeric ligand. In some embodiments, the method further comprises a resolution of skin and liver GvHD within 24 hours after administration of the multimeric ligand. In some embodiments the cells are therapeutic cells and are transduced or transfected with a second nucleic acid that encodes a second heterologous protein. In some embodiments, the therapeutic cells are transduced with a heterologous gene that expresses a chimeric antigen receptor. In some embodiments, the therapeutic cells are transduced with a heterologous gene that expresses a modified TGF-beta receptor. In some embodiments, the therapeutic cells are transduced with the heterologous gene before, at the same time as, or after being transduced with the nucleic acid encoding the chimeric protein comprising a multimerization region and a caspase- 9 polypeptide. Also provided is a method for administering donor T cells to a human patient, comprising administering a transduced or transfected T cell of the present application to a human patient, wherein the cells are non-allodepleted human donor T cells. In some embodiments, the therapeutic cells are administered to a subject having a non-malignant disorder, or where the subject has been diagnosed with a non-malignant disorder, such as, for example, a primary immune deficiency disorder (for example, but not limited to, Severe Combined Immune Deficiency (SCID), Combined Immune Deficiency (CID), Congenital T-cell Defect / Deficiency, Common Variable Immune Deficiency (CVID), Chronic Granulomatous Disease, IPEX (Immune deficiency, polyendocrinopathy, enteropathy, X-linked) or IPEX-like, Wiskott-Aldrich Syndrome, CD40 Ligand Deficiency, Leukocyte Adhesion Deficiency, DOCK 8 Deficiency, IL-10 Deficiency / IL-10 Receptor Deficiency, GATA 2 deficiency, X-linked lymphoproliferative disease (XLP), Cartilage Hair Hypoplasia, and the like), Hemophagocytosis Lymphohistiocytosis (HLH) or other hemophagocytic disorders, Inherited Marrow Failure Disorders (such as, for example, but not limited to, Shwachman Diamond Syndrome, Diamond Blackfan Anemia, Dyskeratosis Congenita, Fanconi Anemia, Congenital Neutropenia, and the like), Hemoglobinopathies (such as, for example, but not limited to, Sickle Cell Disease, Thalassemia, and the like), Metabolic Disorders (such as, for example, but not limited to, Mucopolysaccharidosis, Sphingolipidoses, and the like), or an Osteoclast disorder (such as, for example, but not limited to Osteopetrosis). The therapeutic cells may be, for example, any cell administered to a patient for a desired therapeutic result. The cells may be, for example, T cells, natural killer cells, B cells, macrophages, peripheral blood cells, hematopoietic progenitor cells, bone marrow cells, or tumor cells. The modified caspase-9 polypeptide can also be used to directly kill tumor cells. In one application, vectors comprising polynucleotides coding for the inducible modified caspase-9 polypeptide would be injected into a tumor and after 10-24 hours (to permit protein expression), the ligand inducer, such as, for example, AP1903, would be administered to trigger apoptosis, causing the release of tumor antigens to the microenvironment. To further improve the tumor microenvironment to be more immunogenic, the treatment may be combined with one or more adjuvants (e.g., IL-12, TLRs, IDO inhibitors, etc.). In some embodiments, the cells may be delivered to treat a solid tumor, such as, for example, delivery of the cells to a tumor bed. In some embodiments, a polynucleotide encoding the chimeric caspase-9 polypeptide may be administered as part of a vaccine, or by direct delivery to a tumor bed, resulting in expression of the chimeric caspase-9 polypeptide in the tumor cells, followed by apoptosis of tumor cells following administration of the ligand inducer. Methods of killing tumor cells in vivo using DNA therapy and intratumor vaccines are discussed in, for example, Xie X. et al, Cancer Res 61, 6795-6804 (2001) and Nikitina, E., et al, Cancer Res 65: 4309-4319 (2005). Thus, also provided in some embodiments are nucleic acid vaccines, such as DNA vaccines, wherein the vaccine comprises a nucleic acid comprising a polynucleotide that encodes an inducible, or modified inducible caspase- 9 polypeptide of the present application. The vaccine may be administered to a subject, thereby transforming or transducing target cells in vivo. The ligand inducer is then administered following the methods of the present application. In some embodiments, the modified caspase-9 polypeptide is a truncated modified caspase-9 polypeptide. In some embodiments, the modified caspase-9 polypeptide lacks the Caspase recruitment domain. In some embodiments, the caspase-9 polypeptide comprises the amino acid sequence of SEQ ID NO: 9, or a fragment thereof, or is encoded by the nucleotide sequence of SEQ ID NO: 8, or a fragment thereof. In some embodiments, the methods further comprise administering a multimeric ligand that binds to the multimeric ligand binding region. In some embodiments, the multimeric ligand binding region is selected from the group consisting of FKBP, cyclophilin receptor, steroid receptor, tetracycline receptor, heavy chain antibody subunit, light chain antibody subunit, single chain antibodies comprised of heavy and light chain variable regions in tandem separated by a flexible linker domain, and mutated sequences thereof. In some embodiments, the multimeric ligand binding region is an FKBP12 region. In some embodiments, the multimeric ligand is an FK506 dimer or a dimeric FK506-like analog ligand. In some embodiments, the multimeric ligand is AP1903. In some embodiments, the multimeric ligand is administered to treat graft versus host disease. In some embodiments, the patient exhibits graft versus host disease symptoms before the multimeric ligand is administered. In some embodiments, the patient exhibits one or more Stage 0 graft versus host disease symptoms. In some embodiments, the patient exhibits one or more Stage 1 graft versus host disease symptoms. In some embodiments, the patient exhibits one or more Stage 2 graft versus host disease symptoms. In some embodiments, the patient exhibits one or more Stage 3 graft versus host disease symptoms. In some embodiments, the patient exhibits one or more Stage 4 graft versus host disease symptoms. In some embodiments, more than one dose of the multimeric ligand is administered. In some embodiments, after administration of the multimeric ligand, the number of alloreactive T cells is reduced. In some embodiments, the number of alloreactive T cells is reduced by from about 60% to 99%, about 70% to 95%, from 80% to 90% or about 90% or more after administration of the multimeric ligand. In some embodiments, after administration of the multimeric ligand, donor T cells survive in the patient that are able to expand and are reactive to viruses and fungi. In some embodiments, after administration of the multimeric ligand, donor T cells survive in the patient that are able to expand and are reactive to tumor cells in the patient. In embodiments, the present disclosure provides: - use of a multimeric ligand for controlling survival of transplanted therapeutic cells in a subject; wherein, the transplanted therapeutic cells comprise a nucleic acid that encodes a chimeric polypeptide comprising (a) a multimerization region and (b) a caspase-9 polypeptide or a modified caspase-9 polypeptide, wherein (i) the caspase-9 polypeptide or the modified caspase-9 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9; (ii) the modified caspase-9 polypeptide differs from SEQ ID NO: 9 by at least one amino acid substitution selected from the group consisting of S144A, S144D, Y153A, Y153F, S183A, S195A, S196A, S196D, S307A, D315A, A316G, T317A, T317C, T317E, T317S, P318A, F319A, F319W, F326K, D327G, D327K, D327R, Q328K, Q328R, L329E, L329G, L329K, D330A, D330E, D330G, D330N, D330S, D330V, A331K, C403A, C403S, C403T, F404T, F404W, F404Y, N405A, N405F, N405Q, N405T, F406A, F406T, F406W, F406Y, G402A, G402I, G402Q, G402Y, C403P, F404A, F404S, and F406L; (iii) the modified caspase-9 polypeptide has a reduced IC<com:Sub>50< / com:Sub> and an elongated dose response curve in response to the multimeric ligand compared to a caspase-9 polypeptide that is not modified; and wherein the multimeric ligand binds to the multimerization region in an amount effective to kill less than 50% of transplanted therapeutic cells that express the caspase-9 polypeptide or the modified caspase-9 polypeptide; and - use of a multimeric ligand for controlling survival of transplanted therapeutic cells in a subject; wherein the transplanted therapeutic cells comprise, (a) a first subset of therapeutic cells that comprise a nucleic acid that encodes a chimeric polypeptide comprising a multimerization region and a first caspase-9 polypeptide, wherein the first caspase-9 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9; (b) a second subset of the therapeutic cells with a nucleic acid that encodes a chimeric polypeptide comprising a multimerization region and a second caspase-9 polypeptide, wherein (i) the second caspase-9 polypeptide is a modified caspase-9 polypeptide and comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9, and the modified caspase-9 polypeptide differs from SEQ ID NO: 9 by at least one amino acid substitution selected from the group consisting of S144A, S144D, Y153A, Y153F, S183A, S195A, S196A, S196D, S307A, D315A, A316G, T317A, T317C, T317E, T317S, P318A, F319A, F319W, F326K, D327G, D327K, D327R, Q328K, Q328R, L329E, L329G, L329K, D330A, D330E, D330G, D330N, D330S, D330V, A331K, C403A, C403S, C403T, F404T, F404W, F404Y, N405A, N405F, N405Q, N405T, F406A, F406T, F406W, F406Y, G402A, G402I, G402Q, G402Y, C403P, F404A, F404S, and F406L; (ii) the first caspase-9 polypeptide and the second modified caspase-9 polypeptide comprise different amino acid sequences and have different basal activities; (iii) the second modified caspase-9 polypeptide has a reduced IC<com:Sub>50< / com:Sub> and an elongated dose response curve in response to the multimeric ligand compared to the first caspase-9 polypeptide that is not modified; and wherein the multimeric ligand binds to the multimerization region in an amount effective to kill more of the first subset of therapeutic cells than the second subset of therapeutic cells. Certain embodiments are described further in the following description, examples, claims and drawings. Brief Description of the Drawings The drawings illustrate embodiments of the technology and are not limiting. For clarity and ease of illustration, the drawings are not made to scale and, in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments. FIG. 1A illustrates various iCasp9 expression vectors as discussed herein. FIG. 1B illustrates a representative western blot of full length and truncated caspase-9 protein produced by the expression vectors shown in FIG. 1A. FIGS. 2A-2D graphically present results of experiments performed to evaluate the effect of expression of iCasp9 expression constructs on the phenotype of cells transduced with various iCasp9 expression vectors. FIG. 2A illustrates levels of cell surface markers in transduced and nontransduced cells. FIG. 2B illustrates levels of secretion of Th1 and Th2 type cytokines upon antigen stimulation in transduced and nontransduced cells. FIG. 2C illustrates levels of cytolytic activity against autologous EVB-transformed lymphoblastoid B-cell line (LCL), HLA-mismatched LCL, and HSB-2 in transduced and nontransduced cells. FIG. 2D illustrates the persistence of antigen dependence on iCasp9 transduced cell lines. Note the steady decline of T cells after antigen stimulation is discontinued. Further discussion of experimental conditions and results are presented in the Examples. FIGS. 3A-3D illustrate the results of various experiments performed to determine the efficacy of a chemical inducer of dimerization (CID), in cells expressing iCasp9 expression constructs. FIG. 3A illustrates FACS plots of cells after treatment with CID or carrier. FACS plots are presented for unselected cells (top row of FIG. 3A) and cells selected for high GFP expression (bottom row of FIG. 3A). FIG. 3B illustrates the results of overnight treatment of iCasp9 transduced cells with CID. The treated panel shows cells exhibiting characteristics of apoptosis. FIG. 3C illustrates the results of CID treated and untreated cells stained for annexin-V and 7-AAD. FIG. 3D shows a dose response curve for the CID AP20187. Further discussion of experimental conditions and results are presented in the Examples. FIGS. 4A-4C illustrate the results of various experiments performed to measure the correlation between transgene expression level and function of iCasp9. FIG. 4A show the results of cell population selection based on GFP expression. FIG. 4B illustrates the results of cells treated overnight with CID treated and stained for annexin-V and 7-AAD. FIG. 4C shows the results of selected T cells that were mixed 1:1 with non-transduced T-cells and incubated with 10 nM CID following antigenic stimulation. Indicated is the percentage of residual GFP-positive T-cells on day 7. Further discussion of experimental conditions and results are presented in the Examples. FIGS. 5A-5C illustrate the results of various experiments comparing the functionality of iFas and iCasp9 in T cells. FIG. 5A illustrates the results of cells transduced with an iFas or iCasp9 expression construct and sorted according to GFP expression. FIG. 5B illustrates the results of GFP expression measurements after treatment with CID. FIG. 5C shows the results of expression studies performed in the human derived cell lines Jurkat and MT-2. The cell lines were stained with annexin-V and 7-AAD. Further discussion of experimental conditions and results are presented in the Examples. FIG. 6 graphically illustrates the function of iCasp9 when co-expressed with II-2. FIG. 7 graphically illustrates the function of iCasp9 in vivo. Further discussion of experimental conditions and results are presented in the Examples. FIG. 8A illustrates the structure of the iCasp9 expression construct SFG.iCasp9.2A.ΔCD19. FIG. 8B illustrates the protocol used to produce the cell product expression iCasp9 in allodepleted cells. Further discussion of experimental conditions and results are presented in the Examples. FIG. 9 graphically illustrates that allodepleted cells could be successfully expanded following transduction. FIG. 10 shows that cells transduced with the suicide gene construct could be enriched to high purity by CD19 immunomagnetic selection. Further discussion of experimental conditions and results are presented in the Examples. FIGS. 11A-11C illustrate the results of various experiments performed to show that gene modified allodepleted cells retain their anti-viral repertoire and functionality. FIG. 11A shows the interferon-y secretion in response to viral antigens as assessed by ELISpot. FIG. 11B shows the results of a cytotoxicity assay after allodepleted cells were stimulated with EBV-LCLs. FIG. 11C illustrates the frequency of T cells specific for HLA-B8-RAKFKQLL, an epitope from an EBV lytic antigen (BZLF1). FIGS. 12A and 12B illustrate the results of various experiments performed to show that regulatory T cells could be isolated from gene modified end product cells despite initial allodepletion using CD25 immunotoxin. FIG. 12A shows the levels of Foxp3 expression. FIG. 12B illustrates the results of the functional assay performed to show that addition of CD4<com:Sup>+< / com:Sup> / CD25<com:Sup>+< / com:Sup> gene modified depleted cells significantly reduced cell proliferation. Further discussion of experimental conditions and results are presented in the Examples. FIGS. 13A-13CA and 13CB illustrate the results of various experiments performed to show that gene modified allodepleted cells are rapidly and efficiently eliminated by AP20187, and that transgene expression and killing efficiency diminished with extended culture, and could be restored upon T cell reactivation. FIG. 13A shows representative FACS analysis of cells stained with annexin-V and 7-AAD. FIG. 13B graphically illustrates the results of reactivation of T cells on killing when AP20187 is administered. FIG. 13CA and 13CB show representative FACS plots showing the effect of extended culture and T cell activation on suicide gene function. Further discussion of experimental conditions and results are presented in the Examples. FIGS. 14A and 14B illustrate the results of various experiments performed to show that viral- specific T cells are partially retained after treatment of allostimulated cells with dimerizer. FIG. 14A shows the results for EBV-specific T cells. FIG. 14B shows the results for CMV-specific T cells. Cells were quantified by pentamer analysis before allostimulation, after allostimulation and after treatment of allostimulated cells with dimerizer. Further discussion of experimental conditions and results are presented in the Examples. FIGS. 15A and 15B illustrate an analysis of mesenchymal stromal cells (MSCs) from healthy individuals. FIG. 15A shows the mononuclear adherent fraction isolated from bone marrow was homogenously positive for CD73, CD90 and CD105 and was negative for hematopoietic markers. FIG. 15B illustrate analysis showing the cells were able to differentiate into other cell lineages. Further discussion of experimental conditions and results are presented in the Examples. FIGS. 16A and 16B illustrate the results of experiments performed to show that human MSCs are readily transformed with iCasp9-ΔCD19 and maintain their phenotype. FIG. 16A illustrates the percentage of CD19 positive cells (e.g., an indicator of successful transduction of iCasp9) remains substantially constant for more than 2 weeks. FIG. 16B shows that successfully transduced and non-transduced cells retain the characteristic MSC surface phenotype. Further discussion of experimental conditions and results are presented in the Examples. FIGS. 17A and 17B illustrate the results of experiments performed to show that human MSCs expressing iCasp9 are selectively driven to apoptosis in vitro after exposure to the CID. FIG. 17A shows the results of FACS analysis of cells treated with CID for 24 hours. FIG. 17B shows the results of magnetic purification of iCasp9<com:Sup>+< / com:Sup> / CD19<com:Sup>+< / com:Sup> cells. Further discussion of experimental conditions and results are presented in the Examples. FIG. 18 illustrates the results of experiments performed to determine the efficacy of apoptosis and identify apoptosis resistant populations. FIG. 19, panels A-Q illustrate human MSCs expressing iCasp9 stained to highlight specific cell lineages, showing that the transduced cells retain the differentiation potential of unmodified MSCs. Further discussion of experimental conditions and results are presented in the Examples. FIG. 20 graphically illustrates that the differentiated progeny of human MSCs expressing iCasp9 are killed by exposure to CID in vitro. FIGS. 21A-21C illustrate the results of experiments performed to show that human MSCs expressing iCasp9 are selectively killed in vivo after exposure to CID. FIG. 21A shows the results of whole animal imaging. FIG. 21B graphically shows a time course of the killing of iCasp9<com:Sup>+< / com:Sup> cells after exposure to CID. FIG. 21C shows the results of serial examination of animals after subcutaneous inoculation of MSC. Further discussion of experimental conditions and results are presented in the Examples. FIG. 22 shows how the suicide gene product and the CID interact to cause apoptosis. FIG. 23 illustrates an overview of the protocol used for production of suicide gene modified allodepleted cells. FIG. 24 describes the use of immunomagnetic enrichment of iCasp9 expressing allodepleted T cells. FIG. 25 illustrates the iCasp9-ΔCD19 expression construct and the method of transducing cells to harbor the expression construct. Further discussion of experimental conditions and results are presented in the Examples. FIG. 26 shows the effect of CID treatment on gene modified T cells (e.g., iCasp9 expressing cells). FIGs. 27A and 27B provide graphs showing the detection of iCasp9-transduced T cells in the peripheral blood of patients. Fig. 27A: FACS analysis and Fig: 27B: DNA analysis, for iCasp9- transduced T cells (CD3<com:Sup>+< / com:Sup> CD19<com:Sup>+< / com:Sup>, CD4<com:Sup>+< / com:Sup> CD19<com:Sup>+< / com:Sup>, or CD8<com:Sup>+< / com:Sup> CD19<com:Sup>+< / com:Sup>) from four patients receiving cellular therapy following HLA-haploidentical stem cell transplantation for relapsed leukemia. Patients 1, 2, and 4 developed skin / liver GvHD and received a single dose of the dimerizing drug AP1903. FIGS. 28 and 29 graphically illustrate cell lineage expansion of transduced iCasp9 T cells, as indicated by cell surface markers. FIG. 30 provides a graph and photographs of the rapid reversal of GvHD after treatment with the dimerizing drug AP1903. (A) is a graph depicting the normalization of bilirubin concentration in patient 1 within 24 hours post-treatment. (B) provides photographs showing the disappearance of skin rash from patient 2 within 24 hours post treatment. FIGS. 31 and 32 graphically illustrate the onset of acute liver GvHD (grade 2) after iCasp9 T cell expansion. FIG. 32 also pictorially illustrates a patient exhibiting symptoms of GvHD. FIGS 33-34 show the rapid and efficient elimination of iCasp9 T cells after AP1903 (e.g., the CID) is administered to patients. FIGs. 35A-35C provide graphs showing the persistence of drug sensitivity and antiviral function of CD3<com:Sup>+< / com:Sup> / CD19<com:Sup>+< / com:Sup> precursors after treatment with AP1903 in vivo. (A) CD3<com:Sup>+< / com:Sup>CD19<com:Sup>+< / com:Sup> T cells remain within the CD3<com:Sup>+< / com:Sup> population in the peripheral blood 5 months after treatment with AP1903 (patient 2). These CD3<com:Sup>+< / com:Sup> CD19<com:Sup>+< / com:Sup> cells retain sensitivity to AP1903 in vitro as assessed both by reduction of CD3<com:Sup>+< / com:Sup>CD19<com:Sup>+< / com:Sup> cell number on FACS analysis and (B) by quantitative PCR analysis of the icasp9 gene before and after exposure to the dimerizing drug. (C) CD3<com:Sup>+< / com:Sup> CD19<com:Sup>+< / com:Sup> gene-modified T cells collected from patient 2 were responsive to CMV peptide mixtures at 6 days prior to AP1903, but not to negative control surviving peptide mixtures, as shown by the presence of IFN-gamma- positive CD3<com:Sup>+< / com:Sup> CD19<com:Sup>+< / com:Sup> T cells in the CMV-stimulated cultures. Assessment of the recovering CD3<com:Sup>+< / com:Sup> CD19<com:Sup>+< / com:Sup> population at 6 and 14 days after AP1903 infusion to treat GvHD showed the persistence of virus-specific cells in the absence of recurrent GvHD. FIGS. 36-38 graphically illustrate that iCasp9 allodepleted cells are able to expand after AP1903 treatment without signs of GvHD. FIG. 37 shows reconstitution of naïve, central memory and effector memory T cell after AP1903 treatment. FIG. 39 graphically illustrates iCasp9 allodepleted T cell expansion and restoration of donor chimerism. Further discussion of experimental conditions and results are presented in the Examples. FIG. 40 graphically illustrates virus specific T cells pre and post T cell infusion. FIG. 41 graphically illustrates the levels of intracellular IFN-γ production by patient PBMCs in response to aspergillus antigen. FIG. 42 graphically illustrates iCasp9 T cells expansion. Further discussion of experimental conditions and results are presented in the Examples. FIG. 43 graphically illustrates the portion of the expression construct coding for the chimeric iCaspase9 and CD19 polypeptides. FIG. 44 graphically illustrates a protein structure of a caspase-9 polypeptide. To modify the basal signaling, site-directed mutagenesis was done on residues previously reported to be crucial in homo-dimerization (G402-C-F-N-F406), proteolysis and interaction with XIAP-BIR3 domain (pink), the inhibitor of caspase-9 (gray) (D315-A-P-F319, D330-A-I-S-S334), and phosphorylation sites on caspase-9. The crystallography is based on 1nw9 (RCSB Protein Data Bank). Sixty-five iCasp9 mutants were tested, and promising candidates with lower basal activity included $183A, $196D, D330A, and N405Q. FIG 45 provides an analysis of SEAP assays. To examine both basal signaling and AP1903 induced activity, 106 early-passage HEK293T / 16 cells were co-transfected with various amount of wildtype Caspase and 500 ng of an expression plasmid that uses an SRα promoter to drive SEAP, a marker for cell viability. Following manufacturer's suggestions, 1 mL of IMDM+10% FBS without antibiotics was added to each mixture. 1000-ul of the mixture was seeded onto each well of a 96- well plate. 100-ul of AP1903 was added at least three hours post-transfection. After addition of AP1903 for at least 24 hours, 100-ul of supernatant was transferred to a 96-well plate and heat denatured at 68 C for 30 minutes to inactivate endogenous alkaline phosphatases. For the assay, 4-methylumbelliferyl phosphate substrate was hydrolyzed by SEAP to 4-methylumbelliferon, a metabolite that can be excited with 364 nm and detected with an emission filter of 448 nm. Since SEAP is used as a marker for cell viability, reduced SEAP reading corresponds with increased icaspase-9 activities. Thus, a higher SEAP reading in the absence of AP1903 would indicate lower basal activity. Desired caspase mutants would have diminished basal signaling with increased sensitivity (i.e., lower IC<com:Sub>50< / com:Sub>) to AP1903. The goal of the study is to reduce basal signaling without significantly impairing IC<com:Sub>50< / com:Sub>. FIGs. 46A-46C graphically illustrate data related to basal and AP1903-induced signaling of various chimeric modified caspase-9 polypeptides. (46A) SEAP assay of HEK293 / 16 cells transiently transfected with 1 µg of DNA coding for chimeric modified caspase-9 polypeptides and 0.5 µg of pSH1-kSEAP per million HEK293 cells, 72 hours post-transfection. iCasp9 D330A, N405Q, and D330A-N405Q double mutant all showed lower basal signaling. (46B) HEK293 / 16 cells transfected with 2 µg of DNA coding for chimeric modified caspase-9 polypeptides along with 0.5 μg pSH1-kSEAP per million HEK293 cells. (46C) Summary of estimated AP1903 IC<com:Sub>50< / com:Sub>s of chimeric modified caspase-9 polypeptides. All mutations adversely increased IC<com:Sub>50< / com:Sub> to AP1903. Data points are the average of two wells, and the data shown is representative of two independent experiments. FIGs. 47A-47B include photographs of Western blots analyzing protein expression and proteolysis of chimeric wild type (unmodified) caspase-9 polypeptides and chimeric modified caspase-9 polypeptides. (47A) Western blot of HEK293T / 16 cells transiently transfected with 1 or 2 μg of pSH1-iCasp9 WT, D330A, N405Q, or D330A-N405Q double mutant 72 hours post-transfection. 33 μg of protein lysates were loaded per lane in both blots. The blots were labeled with 1:1000 diluted rabbit anti-human caspase-9 polyclonal antibody targeting residues 299-318 of human caspase-9 to detect both unprocessed and p30 cleavage products. iCasp9 D330A, N405Q, and D330A-N405Q were expressed at similar or higher levels than wild-type iCasp9. (47B) Labeling of stripped blots with anti-actin polyclonal antibody showed equivalent amount of protein loaded in 4A. FIG. 48 provides a graph of a theoretical dose-response curve of an attenuated, titratable modified caspase polypeptide. The left side approximates the typical dose-response curve for a caspase-9 polypeptide that is not modified, with <com:Math><semantics>IC50∼10<annotation encoding="application / x-tex">IC_{50} \sim 10< / annotation>< / semantics>< / com:Math> pM. The right side shows a hypothetical modified caspase-9 polypeptide with both reduced IC<com:Sub>50< / com:Sub> and an elongated dose-response curve. Regardless of extended dose-response curve of hypothetical next-generation inducible CaspaCIDe, both polypeptides could allow modulated, titratable elimination of cells, allowing physician-directed adjustment of cell death, although in different dosage ranges. FIG. 49 (A-B) provides the results of a dose-escalation study in normal male volunteers. Healthy volunteers were infused with the indicated doses of AP1903 and at various time points, serum levels of AP1903 were measured using HPLC analysis. The data show that peak levels of AP1903 can be reliably titrated over about 2logs, with close to Cmax levels at each dose reached within 30 min. FIGs. 50A-50B provide graphs of dose response curves illustrating that modifications in the Caspase9 dimer interface shifts the dose-response curve. (50A) To examine both basal signaling and AP1903-induced CaspaCIDe activity, 10<com:Sup>6< / com:Sup> early-passage HEK293T / 17 cells were co- transfected with 2 µg of inducible caspase variants along with 500 ng of an expression plasmid using an SRa promoter to transcriptionally regulate SEAP, a surrogate marker for cell viability. 200- μl of the transfection mixture containing plasmids, GeneJammer and HEK293T / 17 cells in IMDM + 10% FBS (without antibiotics) was seeded into each well of a 96-well plate. To induce caspase activity, 22 µl of serially diluted AP1903 was added 24 hours post-transfection. (50B) To examine basal and AP1903 induced signaling, 100 µl of supernatant was harvested 48 hours post-treatment and heat-denatured at 68 °C for 1 hour to inactivate endogenous alkaline phosphatases. For the assay, 4-methylumbelliferyl phosphate (4-MUP) substrate was hydrolyzed by SEAP to 4- methylumbelliferon, a metabolite with peak excitation at 364 nm and peak emission at 448 nm. Since SEAP is used as a surrogate marker for cell viability, a reduced SEAP reading corresponds to increased caspase-9 activity. All of the caspase mutants shown revealed diminished basal signaling and higher IC<com:Sub>50< / com:Sub>s for AP1903. Caspase F406T showed the highest IC<com:Sub>50< / com:Sub>, followed by F404Y, F404W, and Caspase 10 (ISAQT), and then N405Q. (50C) Summary of estimated IC50s for AP1903 of CaspaCIDe-2.0 candidates. All the mutants showed increased SEAP activity and IC<com:Sub>50< / com:Sub> to AP1903. The data points represent averages of two wells. The IC<com:Sub>50< / com:Sub> values were determined via four parameter non-linear regression curve fitting in GraphPad Prism 6. FIG. 51 provides an IC<com:Sub>50< / com:Sub> chart for AP1903 dosages corresponding to the various modified caspase-9 polypeptides. FIGs. 52A and 52B: Basal and AP1903-induced signaling of top modified caspase mutants. (52A) SEAP assay of HEK293 / 16 transiently transfected with 2 ug of mutant caspase polypeptide and 0.5 ug of pSH1-kSEAP per million HEK293 72 hours post-transfection. iCASP-9 F404Y, F404W, N405Q, and F406T all show lower basal signaling than WT iCaspase9. (52B) Summary of the basal activity and estimated <com:Math><semantics>IC50<annotation encoding="application / x-tex">IC_{50}< / annotation>< / semantics>< / com:Math> of caspase mutants to AP1903. All mutations shift the <com:Math><semantics>IC50<annotation encoding="application / x-tex">IC_{50}< / annotation>< / semantics>< / com:Math> to AP1903. The data points reflect the average of two wells, and the data shown is representative of two experiments. FIGs. 53A-53B: Basal and AP1903 induced signaling of top modified caspase mutants. (53A) SEAP assay of HEK293 / 16 transiently transfected with 1 or 2 ug of mutant caspase polypeptide and 0.5 ug of pSH1-kSEAP per million HEK293 72 hours post-transfection. iCASP-9 D330A, N405Q, and D330A-N405Q double mutant all showed lower basal signaling compared to the wild- type caspase-9 (dash line). (53B) Summary of estimated <com:Math><semantics>IC50<annotation encoding="application / x-tex">IC_{50}< / annotation>< / semantics>< / com:Math> of caspase mutant polypeptides to AP1903. N405Q adversely increased IC<com:Sub>50< / com:Sub> to AP1903. Combining D330A to N405Q failed to improve IC<com:Sub>50< / com:Sub>. The data points were averages of triplicates, and the data shown is representative of seven experiments. FIG. 54 provides a graph of the SEAP assay showing the decrease in basal activity observed with the T317A and T317S mutants. T317A and T317S mutations are likely to reduce XIAP binding, which would be expected to increase basal signaling, the opposite of what was observed. FIG. 55 provides a bar chart of a SEAP assay of the T317A and T317S mutants. Although T317A and T317S have lower basal activity, they are almost equally sensitive to AP1903 as wildtype caspase-9, making these good candidates for new mutants. FIG. 56: Basal and AP1903-induced activation of D330A variants. SEAP assay of transiently transfected HEK293 / 16 cells with 1 or 2 ug of mutant caspase polypeptides and 0.5 ug of pSH1- kSEAP per million HEK293 cells, 72 hours post-transfection. Normalized data based on 2 ug of each expression plasmid (including WT) are mixed with normalized data from 1 ug-based transfections. iCasp9-D330A, -D330E, and -D330S showed statistically lower basal signaling than wild type caspase-9. FIG. 57: Western blot of HEK293T / 16 cells transiently transfected with 2 ug of pSH1-iCasp9 WT, D330A, D330E, D330N, D330V, D330G, and D330S, 72 hours post-transfection. The blots were labeled with 1:1000 diluted rabbit polyclonal anti-"2A" peptide that recognizes the 2A sequence, derived from several picornaviruses. iCasp9-D330A, -D330E, and -D330G were expressed at similar or higher levels than the wild-type iCasp9. Impaired cleavage in response to AP1903 was observed. * annotates SuperSignal MW Protein ladders (Thermo-Fisher Scientific) and a annotates Precision Plus Protein Dual Color Standards (Bio-Rad). FIG. 58: Effects of various caspase mutations on viral titers derived from PG13 packaging cells cross-transduced with VSV-G envelope-based retroviral supernatants. To examine the effect of CaspaCIDe-derived basal signaling on retrovirus master cell line production, retrovirus packaging cell line, PG13, was cross-transduced five times with VSV-G-based retroviral supernatants in the presence of 4 µg / ml transfection-enhancer, polybrene. CaspaCIDe-transduced PG13 cells were subsequently stained with PE-conjugated anti-human CD19 antibody, as an indication of transduction. CaspaCIDe-D330A, -D330E, and -N405Q-transduced PG13 cells showed enhanced CD19 mean fluorescence intensity (MFI), indicating higher retroviral copy numbers, implying lower basal activity. To more directly examine the viral titer of the PG13 transductants, HT1080 cells were treated with viral supernatant and 8 ug / ml polybrene. The enhanced CD19 MFIs of iCasp9- D330A, -N405Q, and -D330E transductants vs WT iCasp9 in PG13 cells are positively correlated with higher viral titers, as observed in HT1080 cells. Due to the initially low viral titers (approximately 1E5 transduction units (TU) / ml), no differences in viral titers were observed in the absence of HAT treatment to increase virus yields. Upon HAT media treatment, PG13 cells transduced with CaspaClDe-D330A, -N405Q, or -D330E demonstrated higher viral titers. Viral titer (transducing units) is calculated with the formula: Viral titer = (# cells on the day of transduction) * (% CD19<com:Sup>+< / com:Sup>) / Volume of supernatant (ml). In order to further investigate the effect of CaspaClDe mutants with lower basal activity, individual clones (colonies) of CaspaClDe-transduced PG13 cells were selected and expanded. CaspaCIDe-N405Q clones with higher CD19 MFIs than the other cohorts were observed. FIG. 59: AP1903 dose-dependent elimination of T cells transduced with iCasp9 mutants. Primary T cells from healthy donors (n=6) were transduced with retrovirus encoding mutant or wild-type iCasp9 and the ΔCD19 cell surface marker. Following transduction, iCasp9-transduced T cells were purified using CD19-microbeads and a magnetic column. T cells were then exposed to AP1903 (0-10 nM) and measured for CD3<com:Sup>+< / com:Sup>CD19<com:Sup>+< / com:Sup>T cells by flow cytometry after 24 hours. FIG. 60: iCasp9-D330A mutant demonstrates improved AP1903-dependent cytotoxicity in transduced T cells. Primary T cells from healthy donors (n=6) were transduced with retrovirus encoding mutant or wild-type iCasp9 or iCasp9-D330A, and the ΔCD19 cell surface marker. Following transduction, iCasp9-transduced T cells were purified using CD19-microbeads and a magnetic column. T cells were then exposed to AP1903 (0-100 nM) and measured for CD3+CD19+ T cells by flow cytometry after 24 hours. FIG. 61: iCasp9-D330A mutant demonstrates improved AP1903-dependent cytotoxicity in transduced T cells. Primary T cells from healthy donors (n=6) were transduced with retrovirus encoding mutant or wild-type iCasp9 or iCasp9-D330A, and the ΔCD19 cell surface marker. Following transduction, iCasp9-transduced T cells were purified using CD19-microbeads and a magnetic column. T cells were then exposed to AP1903 (0-100 nM) and measured for CD3<com:Sup>+< / com:Sup>CD19<com:Sup>+< / com:Sup> T cells by flow cytometry after 24 hours. The IC<com:Sub>50< / com:Sub> of iCasp9-D330A was significantly lower (p=0.002) than wild-type iCasp9. FIG. 62: D330 family members demonstrate similar AP1903-dependent cytotoxicity as D330A in transduced T cells. Primary T cells from healthy donors (n=2) were transduced with retrovirus encoding D330 mutants or wild-type iCasp9 and the ΔCD19 cell surface marker. Following transduction, iCasp9-transduced T cells were purified using CD19-microbeads and a magnetic column. T cells were then exposed to AP1903 (0-100 nM) and measured for CD3<com:Sup>+< / com:Sup>CD19<com:Sup>+< / com:Sup>T cells by flow cytometry after 24 hours. FIG. 63: iCasp9 mutants with lower sensitivity to AP1903 still control T cell proliferation in vitro. Activated T cells were transduced with retrovirus containing iCasp9 mutants and treated with AP1903 (arrow) on days 0 and 4 and subsequently enumerated for 10 days. Both wild-type, D330A and other iCasp9 mutants halted T cell proliferation and decreased T cell survival after 10 days. FIG. 64: Improved transgene expression of N405Q mutants improves AP1903-dependent cytotoxicity in transduced T cells. T cells were transduced with RD114 pseudotyped retrovirus encoding iCasp9 wild-type, N405Q, codon-optimized N405Q and Fv.Fv'.N405Q and then treated with a varying dose of AP1903 (0-100 nM). After 24 hours, CD3<com:Sup>+< / com:Sup>CD19<com:Sup>+< / com:Sup> T cells were measured by flow cytometry. Percent remaining was normalized to the frequency of CD3<com:Sup>+< / com:Sup>CD19<com:Sup>+< / com:Sup> T cells without AP1903. FIG. 65: AP1903 dose-dependent elimination in vivo of T cells transduced with wild-type iCasp9. T cells were transduced with SFG-iCasp9-2A-\DOD19 retrovirus and injected i.v. into immune deficient mice (NSG). After 24 hours, mice were injected i.p. with AP1903 (0-5 mg / kg). After an additional 24 hours, mice were sacrificed and lymphocytes from the spleen (A) and peripheral blood (B) were isolated and analyzed by flow cytometry for the frequency of human CD3*CD19*T cells. FIG. 66: AP1903 dose-dependent elimination in vivo of T cells transduced with D330E iCasp9. T cells were transduced with SFG-iCasp9-D330E-2A-ΔCD19 retrovirus and injected i.v. into immune deficient mice (NSG). After 24 hours, mice were injected i.p. with AP1903 (0-5 mg / kg). After an additional 24 hours, mice were sacrificed and lymphocytes from the spleen (A) were isolated and analyzed by flow cytometry for the frequency of human CD3<com:Sup>+< / com:Sup>CD19<com:Sup>+< / com:Sup> T cells. This shows that iCasp9-D330E demonstrates a similar in vivo cytotoxicity profile in response to AP1903 as wild- type iCasp9. Detailed Description As used herein, the use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." Still further, the terms "having", "including", "containing" and "comprising" are interchangeable and one of skill in the art is cognizant that these terms are open ended terms. The term "allogeneic" as used herein, refers to HLA or MHC loci that are antigenically distinct. Thus, cells or tissue transferred from the same species can be antigenically distinct. Syngeneic mice can differ at one or more loci (congenics) and allogeneic mice can have the same background. The term "antigen" 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. The term "cancer" as used herein is defined as a hyperproliferation of cells whose unique trait— loss of normal controls—results in unregulated growth, lack of differentiation, local tissue invasion, and metastasis. Examples include but are not limited to, melanoma, non-small cell lung, small-cell lung, lung, hepatocarcinoma, leukemia, retinoblastoma, astrocytoma, glioblastoma, gum, tongue, neuroblastoma, head, neck, breast, pancreatic, prostate, renal, bone, testicular, ovarian, mesothelioma, cervical, gastrointestinal, lymphoma, brain, colon, sarcoma or bladder. Donor: The term "donor" refers to a mammal, for example, a human, that is not the patient recipient. The donor may, for example, have HLA identity with the recipient, or may have partial or greater HLA disparity with the recipient. Haploidentical: The term "haploidentical" as used with reference to cells, cell types and / or cell lineages, herein refers to cells sharing a haplotype or cells having substantially the same alleles at a set of closely linked genes on one chromosome. A haploidentical donor does not have complete HLA identity with the recipient, there is a partial HLA disparity. Blood disease: The terms "blood disease", "blood disease" and / or "diseases of the blood" as used herein, refers to conditions that affect the production of blood and its components, including but not limited to, blood cells, hemoglobin, blood proteins, the mechanism of coagulation, production of blood, production of blood proteins, the like and combinations thereof. Non-limiting examples of blood diseases include anemias, leukemias, lymphomas, hematological neoplasms, albuminemias, haemophilias and the like. Bone marrow disease: The term "bone marrow disease" as used herein, refers to conditions leading to a decrease in the production of blood cells and blood platelets. In some bone marrow diseases, normal bone marrow architecture can be displaced by infections (e.g., tuberculosis) or malignancies, which in turn can lead to the decrease in production of blood cells and blood platelets. Non-limiting examples of bone marrow diseases include leukemias, bacterial infections (e.g., tuberculosis), radiation sickness or poisoning, apnocytopenia, anemia, multiple myeloma and the like. T cells and Activated T cells (include that this means CD3<com:Sup>+< / com:Sup> cells): T cells (also referred to as T lymphocytes) belong to a group of white blood cells referred to as lymphocytes. Lymphocytes generally are involved in cell-mediated immunity. The "T" in "T cells" refers to cells derived from or whose maturation is influenced by the thymus. T cells can be distinguished from other lymphocytes types such as B cells and Natural Killer (NK) cells by the presence of cell surface proteins known as T cell receptors. The term "activated T cells" as used herein, refers to T cells that have been stimulated to produce an immune response (e.g., clonal expansion of activated T cells) by recognition of an antigenic determinant presented in the context of a Class II major histo- compatibility (MHC) marker. T-cells are activated by the presence of an antigenic determinant, cytokines and / or lymphokines and cluster of differentiation cell surface proteins (e.g., CD3, CD4, CD8, the like and combinations thereof). Cells that express a cluster of differential protein often are said to be "positive" for expression of that protein on the surface of T-cells (e.g., cells positive for CD3 or CD 4 expression are referred to as CD3<com:Sup>+< / com:Sup> or CD4<com:Sup>+< / com:Sup>). CD3 and CD4 proteins are cell surface receptors or co-receptors that may be directly and / or indirectly involved in signal transduction in T cells. Peripheral blood: The term "peripheral blood" as used herein, refers to cellular components of blood (e.g., red blood cells, white blood cells and platelets), which are obtained or prepared from the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver or bone marrow. Umbilical cord blood: Umbilical cord blood is distinct from peripheral blood and blood sequestered within the lymphatic system, spleen, liver or bone marrow. The terms "umbilical cord blood", "umbilical blood" or "cord blood", which can be used interchangeably, refers to blood that remains in the placenta and in the attached umbilical cord after child birth. Cord blood often contains stem cells including hematopoietic cells. By "obtained or prepared" as, for example, in the case of cells, is meant that the cells or cell culture are isolated, purified, or partially purified from the source, where the source may be, for example, umbilical cord blood, bone marrow, or peripheral blood. The terms may also apply to the case where the original source, or a cell culture, has been cultured and the cells have replicated, and where the progeny cells are now derived from the original source. By "kill" or "killing" as in a percent of cells killed, is meant the death of a cell through apoptosis, as measured using any method known for measuring apoptosis, and, for example, using the assays discussed herein, such as, for example the SEAP assays or T cell assays discussed herein. The term may also refer to cell ablation. Allodepletion: The term "allodepletion" as used herein, refers to the selective depletion of alloreactive T cells. The term "alloreactive T cells" as used herein, refers to T cells activated to produce an immune response in reaction to exposure to foreign cells, such as, for example, in a transplanted allograft. The selective depletion generally involves targeting various cell surface expressed markers or proteins, (e.g., sometimes cluster of differentiation proteins (CD proteins), CD19, or the like, for removal using immunomagnets, immunotoxins, flow sorting, induction of apoptosis, photodepletion techniques, the like or combinations thereof. In the present methods, the cells may be transduced or transfected with the chimeric protein-encoding vector before or after allodepletion. Also, the cells may be transduced or transfected with the chimeric protein- encoding vector without an allodepletion step, and the non-allodepleted cells may be administered to the patient. Because of the added "safety switch" it is, for example, possible to administer the non allo-depleted T cells because an adverse event such as, for example, graft versus host disease, may be alleviated upon the administration of the multimeric ligand. Graft versus host disease: The terms "graft versus host disease" or "GvHD", refer to a complication often associated with allogeneic bone marrow transplantation and sometimes associated with transfusions of un-irradiated blood to immunocompromised patients. Graft versus host disease sometimes can occur when functional immune cells in the transplanted marrow recognize the recipient as "foreign" and mount an immunologic response. GvHD can be divided into an acute form and a chronic form. Acute GVHD (aGVHD) often is observed within the first 100 days following transplant or transfusion and can affect the liver, skin, mucosa, immune system (e.g., the hematopoietic system, bone marrow, thymus, and the like), lungs and gastrointestinal tract. Chronic GVHD (cGVHD) often begins 100 days or later post transplant or transfusion and can attack the same organs as acute GvHD, but also can affect connective tissue and exocrine glands. Acute GvHD of the skin can result in a diffuse maculopapular rash, sometimes in a lacy pattern. Graft versus host disease may be diagnosed as having a particular stage. Alleviating the disease may include, for example, reducing the stage of the disease. For example, after treatment, a patient exhibiting Stage 4 symptoms may exhibit Stage 3, 2, or 1 symptoms, or no symptoms of GvHD. Donor T cell: The term "donor T cell" as used here refers to T cells that often are administered to a recipient to confer anti-viral and / or anti-tumor immunity following allogeneic stem cell transplantation. Donor T cells often are utilized to inhibit marrow graft rejection and increase the success of alloengraftment, however the same donor T cells can cause an alloaggressive response against host antigens, which in turn can result in graft versus host disease (GVHD). Certain activated donor T cells can cause a higher or lower GvHD response than other activated T cells. Donor T cells may also be reactive against recipient tumor cells, causing a beneficial graft vs. tumor effect. Mesenchymal stromal cell: The terms "mesenchymal stromal cell" or "bone marrow derived mesenchymal stromal cell" as used herein, refer to multipotent stem cells that can differentiate ex vivo, in vitro and in vivo into adipocytes, osteoblasts and chondroblasts, and may be further defined as a fraction of mononuclear bone marrow cells that adhere to plastic culture dishes in standard culture conditions, are negative for hematopoietic lineage markers and are positive for CD73, CD90 and CD105. Embryonic stem cell: The term "embryonic stem cell" as used herein, refers to pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo of between 50 to 150 cells. Embryonic stem cells are characterized by their ability to renew themselves indefinitely and by their ability to differentiate into derivatives of all three primary germ layers, ectoderm, endoderm and mesoderm. Pluripotent is distinguished from mutipotent in that pluripotent cells can generate all cell types, while multipotent cells (e.g., adult stem cells) can only produce a limited number of cell types. Inducible pluripotent stem cell: The terms "inducible pluripotent stem cell" or "induced pluripotent stem cell" as used herein refers to adult, or differentiated cells, that are "reprogrammed" or induced by genetic (e.g., expression of genes that in turn activates pluripotency), biological (e.g., treatment viruses or retroviruses) and / or chemical (e.g., small molecules, peptides and the like) manipulation to generate cells that are capable of differentiating into many if not all cell types, like embryonic stem cells. Inducible pluripotent stem cells are distinguished from embryonic stem cells in that they achieve an intermediate or terminally differentiated state (e.g., skin cells, bone cells, fibroblasts, and the like) and then are induced to dedifferentiate, thereby regaining some or all of the ability to generate multipotent or pluripotent cells. CD34<com:Sup>+< / com:Sup> cell: The term "CD34<com:Sup>+< / com:Sup> cell" as used herein refers to a cell expressing the CD34 protein on its cell surface. "CD34" as used herein refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion factor and is involved in T cell entrance into lymph nodes, and is a member of the "cluster of differentiation" gene family. CD34 also may mediate the attachment of stem cells to bone marrow, extracellular matrix or directly to stromal cells. CD34<com:Sup>+< / com:Sup> cells often are found in the umbilical cord and bone marrow as hematopoietic cells, a subset of mesenchymal stem cells, endothelial progenitor cells, endothelial cells of blood vessels but not lymphatics (except pleural lymphatics), mast cells, a sub-population of dendritic cells (which are factor XIIIa negative) in the interstitium and around the adnexa of dermis of skin, as well as cells in certain soft tissue tumors (e.g., alveolar soft part sarcoma, pre-B acute lymphoblastic leukemia (Pre-B-ALL), acute myelogenous leukemia (AML), AML-M7, dermatofibrosarcoma protuberans, gastrointestinal stromal tumors, giant cell fibroblastoma, granulocytic sarcoma, Kaposi's sarcoma, liposarcoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumors, mengingeal hemangiopericytomas, meningiomas, neurofibromas, schwannomas, and papillary thyroid carcinoma). Tumor infiltrating lymphocytes (TILs) refer to T cells having various receptors which infiltrate tumors and kill tumor cells in a targeted manor. Regulating the activity of the TILs using the methods of the present application would allow for more direct control of the elimination of tumor cells. Gene expression vector: The terms "gene expression vector", "nucleic acid expression vector", or "expression vector" as used herein, which can be used interchangeably throughout the document, generally refers to a nucleic acid molecule (e.g., a plasmid, phage, autonomously replicating sequence (ARS), artificial chromosome, yeast artificial chromosome (e.g., YAC)) that can be replicated in a host cell and be utilized to introduce a gene or genes into a host cell. The genes introduced on the expression vector can be endogenous genes (e.g., a gene normally found in the host cell or organism) or heterologous genes (e.g., genes not normally found in the genome or on extra-chromosomal nucleic acids of the host cell or organism). The genes introduced into a cell by an expression vector can be native genes or genes that have been modified or engineered. The gene expression vector also can be engineered to contain 5' and 3' untranslated regulatory sequences that sometimes can function as enhancer sequences, promoter regions and / or terminator sequences that can facilitate or enhance efficient transcription of the gene or genes carried on the expression vector. A gene expression vector sometimes also is engineered for replication and / or expression functionality (e.g., transcription and translation) in a particular cell type, cell location, or tissue type. Expression vectors sometimes include a selectable marker for maintenance of the vector in the host or recipient cell. Developmentally regulated promoter: The term "developmentally regulated promoter" as used herein refers to a promoter that acts as the initial binding site for RNA polymerase to transcribe a gene which is expressed under certain conditions that are controlled, initiated by or influenced by a developmental program or pathway. Developmentally regulated promoters often have additional control regions at or near the promoter region for binding activators or repressors of transcription that can influence transcription of a gene that is part of a development program or pathway. Developmentally regulated promoters sometimes are involved in transcribing genes whose gene products influence the developmental differentiation of cells. Developmentally differentiated cells: The term "developmentally differentiated cells", as used herein refers to cells that have undergone a process, often involving expression of specific developmentally regulated genes, by which the cell evolves from a less specialized form to a more specialized form in order to perform a specific function. Non-limiting examples of developmentally differentiated cells are liver cells, lung cells, skin cells, nerve cells, blood cells, and the like. Changes in developmental differentiation generally involve changes in gene expression (e.g., changes in patterns of gene expression), genetic re-organization (e.g., remodeling or chromatin to hide or expose genes that will be silenced or expressed, respectively), and occasionally involve changes in DNA sequences (e.g., immune diversity differentiation). Cellular differentiation during development can be understood as the result of a gene regulatory network. A regulatory gene and its cis-regulatory modules are nodes in a gene regulatory network that receive input (e.g., protein expressed upstream in a development pathway or program) and create output elsewhere in the network (e.g., the expressed gene product acts on other genes downstream in the developmental pathway or program). The terms "cell," "cell line," and "cell culture" as used herein may be used interchangeably. All of these terms also include their progeny, which are any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. As used herein, the term "icaspase-9 molecule," polypeptide, or protein, is defined as an inducible caspase-9. The term "icaspase-9" embraces icaspase-9 nucleic acids, icaspase-9 polypeptides and / or icaspase-9 expression vectors. The term also encompasses either the natural icaspase-9 nucleotide or amino acid sequence, or a truncated sequence that is lacking the CARD domain. Without indicating that the polypeptide is "modified" by use of the term, or other means, a "caspase-9 polypeptide" is considered to by "wild type." By "wild type" caspase-9 polypeptide in the context of the experimental details provided herein, is meant the caspase-9 polypeptide lacking the CARD domain. As used herein, the term "iCaspase 1 molecule", "iCaspase 3 molecule", or "iCaspase 8 molecule" is defined as an inducible Caspase 1, 3, or 8, respectively. The term iCaspase 1, iCaspase 3, or iCaspase 8, embraces iCaspase 1, 3, or 8 nucleic acids, iCaspase 1, 3, or 8 polypeptides and / or iCaspase 1, 3, or 8 expression vectors, respectively. The term also encompasses either the natural CaspaseiCaspase-1, -3, or -8 nucleotide or amino acid sequence, respectively, or a truncated sequence that is lacking the CARD domain. Modified caspase-9 polypeptides comprise at least one amino acid substitution that affects basal activity or IC<com:Sub>50< / com:Sub>, in a chimeric polypeptide comprising the modified caspase-9 polypeptide. Methods for testing basal activity and IC<com:Sub>50< / com:Sub> are discussed herein. Caspase-9 polypeptides that are not modified do not comprise this type of amino acid substitution. Both modified caspase-9 polypeptides and caspase-9 polypeptides that are not modified may be truncated, for example, to remove the CARD domain. "Function-conservative variants" are proteins or enzymes in which a given amino acid residue has been changed without altering overall conformation and function of the protein or enzyme, including, but not limited to, replacement of an amino acid with one having similar properties, including polar or non-polar character, size, shape and charge. Conservative amino acid substitutions for many of the commonly known non-genetically encoded amino acids are well known in the art. Conservative substitutions for other non-encoded amino acids can be determined based on their physical properties as compared to the properties of the genetically encoded amino acids. Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and can be, for example, at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, as determined according to an alignment scheme. As referred to herein, "sequence similarity" means the extent to which nucleotide or protein sequences are related. The extent of similarity between two sequences can be based on percent sequence identity and / or conservation. "Sequence identity" herein means the extent to which two nucleotide or amino acid sequences are invariant. "Sequence alignment" means the process of lining up two or more sequences to achieve maximal levels of identity (and, in the case of amino acid sequences, conservation) for the purpose of assessing the degree of similarity. Numerous methods for aligning sequences and assessing similarity / identity are known in the art such as, for example, the Cluster Method, wherein similarity is based on the MEGALIGN algorithm, as well as BLASTN, BLASTP, and FASTA. When using any of these programs, the preferred settings are those that results in the highest sequence similarity. The amino acid residue numbers referred to herein reflect the amino acid position in the non- truncated and non-modified caspase-9 polypeptide, for example, that of SEQ ID NO: 9. SEQ ID NO: 9 provides an amino acid sequence for the truncated caspase-9 polypeptide, which does not include the CARD domain. Thus SEQ ID NO: 9 commences at amino acid residue number 135, and ends at amino acid residue number 416, with reference to the full length caspase-9 amino acid sequence. Those of ordinary skill in the art may align the sequence with other sequences of caspase-9 polypeptides to, if desired, correlate the amino acid residue number, for example, using the sequence alignment methods discussed herein. As used herein, the term "cDNA" is intended to refer to DNA prepared using messenger RNA (mRNA) as template. The advantage of using a cDNA, as opposed to genomic DNA or DNA polymerized from a genomic, non- or partially-processed RNA template, is that the cDNA primarily contains coding sequences of the corresponding protein. There are times when the full or partial genomic sequence is used, such as where the non-coding regions are required for optimal expression or where non-coding regions such as introns are to be targeted in an antisense strategy. As used herein, the term "expression construct" or "transgene" is defined as any type of genetic construct containing a nucleic acid coding for gene products in which part or all of the nucleic acid encoding sequence is capable of being transcribed can be inserted into the vector. The transcript is translated into a protein, but it need not be. In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding genes of interest. The term "therapeutic construct" may also be used to refer to the expression construct or transgene. The expression construct or transgene may be used, for example, as a therapy to treat hyperproliferative diseases or disorders, such as cancer, thus the expression construct or transgene is a therapeutic construct or a prophylactic construct. As used herein, the term "expression vector" refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are discussed infra. As used herein, the term "ex vivo" refers to "outside" the body. The terms "ex vivo" and "in vitro" can be used interchangeably herein. As used herein, the term "functionally equivalent," as it relates to caspase-9, or truncated caspase- 9, for example, refers to a caspase-9 nucleic acid fragment, variant, or analog, refers to a nucleic acid that codes for a caspase-9 polypeptide, or a caspase-9 polypeptide, that stimulates an apoptotic response. "Functionally equivalent" refers, for example, to a caspase-9 polypeptide that is lacking the CARD domain, but is capable of inducing an apoptotic cell response. When the term "functionally equivalent" is applied to other nucleic acids or polypeptides, such as, for example, CD19, the 5'LTR, the multimeric ligand binding region, or CD3, it refers to fragments, variants, and the like that have the same or similar activity as the reference polypeptides of the methods herein. As used herein, the term "gene" is defined as a functional protein, polypeptide, or peptide-encoding unit. As will be understood, this functional term includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or are adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. The term "hyperproliferative disease" is defined as a disease that results from a hyperproliferation of cells. Exemplary hyperproliferative diseases include, but are not limited to cancer or autoimmune diseases. Other hyperproliferative diseases may include vascular occlusion, restenosis, atherosclerosis, or inflammatory bowel disease. The term "immunogenic composition" or "immunogen" refers to a substance that is capable of provoking an immune response. Examples of immunogens include, e.g., antigens, autoantigens that play a role in induction of autoimmune diseases, and tumor-associated antigens expressed on cancer cells. The term "immunocompromised" as used herein is defined as a subject that has reduced or weakened immune system. The immunocompromised condition may be due to a defect or dysfunction of the immune system or to other factors that heighten susceptibility to infection and / or disease. Although such a categorization allows a conceptual basis for evaluation, immunocompromised individuals often do not fit completely into one group or the other. More than one defect in the body's defense mechanisms may be affected. For example, individuals with a specific T-lymphocyte defect caused by HIV may also have neutropenia caused by drugs used for antiviral therapy or be immunocompromised because of a breach of the integrity of the skin and mucous membranes. An immunocompromised state can result from indwelling central lines or other types of impairment due to intravenous drug abuse; or be caused by secondary malignancy, malnutrition, or having been infected with other infectious agents such as tuberculosis or sexually transmitted diseases, e.g., syphilis or hepatitis. As used herein, the term "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells presented herein, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. Nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR<com:Sup>TM< / com:Sup>, and the like, and by synthetic means. Furthermore, polynucleotides include mutations of the polynucleotides, include but are not limited to, mutation of the nucleotides, or nucleosides by methods well known in the art. A nucleic acid may comprise one or more polynucleotides. As used herein, the term "polypeptide" is defined as a chain of amino acid residues, usually having a defined sequence. As used herein the term polypeptide is interchangeable with the terms "peptides" and "proteins". As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The term "transfection" and "transduction" are interchangeable and refer to the process by which an exogenous DNA sequence is introduced into a eukaryotic host cell. Transfection (or transduction) can be achieved by any one of a number of means including electroporation, microinjection, gene gun delivery, retroviral infection, lipofection, superfection and the like. As used herein, the term "syngeneic" refers to cells, tissues or animals that have genotypes that are identical or closely related enough to allow tissue transplant, or are immunologically compatible. For example, identical twins or animals of the same inbred strain. Syngeneic and isogeneic can be used interchangeably. The terms "patient" or "subject" are interchangeable, and, as used herein include, but are not limited to, an organism or animal; a mammal, including, e.g., a human, non-human primate (e.g., monkey), mouse, pig, cow, goat, rabbit, rat, guinea pig, hamster, horse, monkey, sheep, or other non-human mammal; a non-mammal, including, e.g., a non-mammalian vertebrate, such as a bird (e.g., a chicken or duck) or a fish, and a non-mammalian invertebrate. As used herein, the term "under transcriptional control" or "operatively linked" is defined as the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. As used herein, the terms "treatment", "treat", "treated", or "treating" refer to prophylaxis and / or therapy. As used herein, the term "vaccine" refers to a formulation that contains a composition presented herein which is in a form that is capable of being administered to an animal. Typically, the vaccine comprises a conventional saline or buffered aqueous solution medium in which the composition is suspended or dissolved. In this form, the composition can be used conveniently to prevent, ameliorate, or otherwise treat a condition. Upon introduction into a subject, the vaccine is able to provoke an immune response including, but not limited to, the production of antibodies, cytokines and / or other cellular responses. In some embodiments, the nucleic acid is contained within a viral vector. In certain embodiments, the viral vector is a retroviral vector. In certain embodiments, the viral vector is an adenoviral vector or a lentiviral vector. It is understood that in some embodiments, the antigen-presenting cell is contacted with the viral vector ex vivo, and in some embodiments, the antigen-presenting cell is contacted with the viral vector in vivo. Hematopoietic Stem Cells and Cell Therapy Hematopoietic stem cells include hematopoietic progenitor cells, immature, multipotent cells that can differentiate into mature blood cell types. These stem cells and progenitor cells may be isolated from bone marrow and umbilical cord blood, and, in some cases, from peripheral blood. Other stem and progenitor cells include, for example, mesenchymal stromal cells, embryonic stem cells, and inducible pluripotent stem cells. Bone marrow derived mesenchymal stromal cells (MSCs) have been defined as a fraction of mononuclear bone marrow cells that adhere to plastic culture dishes in standard culture conditions, are negative for hematopoietic lineage markers and positive for CD73, CD90 and CD105, and able to differentiate in vitro into adipocytes, osteoblasts, and chondroblasts. While one physiologic role is presumed to be the support of hematopoiesis, several reports have also established that MSCs are able to incorporate and possibly proliferate in areas of active growth, such as cicatricial and neoplastic tissues, and to home to their native microenvironment and replace the function of diseased cells. Their differentiation potential and homing ability make MSCs attractive vehicles for cellular therapy, either in their native form for regenerative applications, or through their genetic modification for delivery of active biological agents to specific microenvironments such as diseased bone marrow or metastatic deposits. In addition, MSCs possess potent intrinsic immunosuppressive activity, and to date have found their most frequent application in the experimental treatment of graft-versus-host disease and autoimmune disorders (Pittenger, M. F., et al. (1999). Science 284: 143-147; Dominici, M., et al. (2006). Cytotherapy 8: 315-317; Prockop, D. J. (1997). Science 276: 71-74; Lee, R. H., et al. (2006). Proc Natl Acad Sci U S A 103: 17438- 17443; Studeny, M., et al., (2002). Cancer Res 62: 3603-3608; Studeny, M., et al. (2004). J Natl Cancer Inst 96: 1593-1603; Horwitz, E. M., et al. (1999). Nat Med 5: 309-313; Chamberlain, G., et al., (2007). Stem Cells 25: 2739-2749; Phinney, D. G., and Prockop, D. J. (2007). Stem Cells 25: 2896-2902; Horwitz, E. M., et al. (2002). Proc Natl Acad Sci U S A 99: 8932-8937; Hall, B., et al., (2007). Int J Hematol 86: 8-16; Nauta, A. J., and Fibbe, W. E. (2007). Blood 110: 3499-3506; Le Blanc, K., et al. (2008). Lancet 371: 1579-1586; Tyndall, A., and Uccelli, A. (2009). Bone Marrow Transplant). MSCs have been infused in hundreds of patients with minimal reported side effects. However, follow-up is limited, long term side effects are unknown, and little is known of the consequences that will be associated with future efforts to induce their in vivo differentiation, for example to cartilage or bone, or to genetically modify them to enhance their functionality. Several animal models have raised safety concerns. For instance, spontaneous osteosarcoma formation in culture has been observed in murine derived MSCs. Furthermore, ectopic ossification and calcification foci have been described in mouse and rat models of myocardial infarction after local injection of MSC, and their proarrhythmic potential has also been apparent in co-culture experiments with neonatal rat ventricular myocytes. Moreover, bilateral diffuse pulmonary ossification has been observed after bone marrow transplant in a dog, presumably due to the transplanted stromal components (Horwitz, E. M., et al., (2007). Biol Blood Marrow Transplant 13: 53-57; Tolar, J., et al. (2007). Stem Cells 25: 371-379; Yoon, Y.-S., et al., (2004). Circulation 109: 3154-3157; Breitbach, M., et al. (2007). Blood 110: 1362-1369; Chang, M. G., et al. (2006). Circulation 113: 1832-1841; Sale, G. E., and Storb, R. (1983). Exp Hematol 11: 961-966). In another example of cell therapy, T cells transduced with a nucleic acid encoding a chimeric antigen receptor have been administered to patients to treat cancer (Zhong, X.-S., (2010) Molecular Therapy 18:413-420). For example, T cells expressing a chimeric antigen receptor based on the humanized monoclonal antibody Trastuzumab (Herceptin) has been used to treat cancer patients. Adverse events are possible, however, and in at least one reported case, the therapy had fatal consequences to the patient (Morgan, R.A., et al., (2010) Molecular Therapy 18:843-851). Transducing the cells with a chimeric caspase-9-based safety switch as presented herein, would provide a safety switch that could stop the adverse event from progressing. By "chimeric antigen receptor" or "CAR" is meant, for example, a chimeric polypeptide which comprises a polypeptide sequence that recognizes a target antigen (an antigen-recognition domain) linked to a transmembrane polypeptide and intracellular domain polypeptide selected to activate the T cell and provide specific immunity. The antigen-recognition domain may be a single- chain variable fragment (ScFv), or may, for example, be derived from other molecules such as, for example, a T cell receptor or Pattern Recognition Receptor. The intracellular domain comprises at least one polypeptide which causes activation of the T cell, such as, for example, but not limited to, CD3 zeta, and, for example, co-stimulatory molecules, for example, but not limited to, CD28, OX40 and 4-1BB. The term "chimeric antigen receptor" may also refer to chimeric receptors that are not derived from antibodies, but are chimeric T cell receptors. These chimeric T cell receptors may comprise a polypeptide sequence that recognizes a target antigen, where the recognition sequence may be, for example, but not limited to, the recognition sequence derived from a T cell receptor or an scFv. The intracellular domain polypeptides are those that act to activate the T cell. Chimeric T cell receptors are discussed in, for example, Gross, G., and Eshar, Z., FASEB Journal 6:3370-3378 (1992), and Zhang, Y., et al., PLOS Pathogens 6:1- 13 (2010). It is understood that by "derived" is meant that the nucleotide sequence or amino acid sequence may be derived from the sequence of the molecule. The intracellular domain comprises at least one polypeptide which causes activation of the T cell, such as, for example, but not limited to, CD3 zeta, and, for example, co-stimulatory molecules, for example, but not limited to, CD28, OX40 and 4-1BB. In another example of cell therapy, T cells are modified so that express a non-functional TGF-beta receptor, rendering them resistant to TGF-beta. This allows the modified T cells to avoid the cytotoxicity caused by TGF-beta, and allows the cells to be used in cellular therapy (Bollard, C.J., et al., (2002) Blood 99:3179-3187; Bollard, C.M., et al., (2004) J. Exptl. Med. 200:1623-1633). However, it also could result in a T cell lymphoma, or other adverse effect, as the modified T cells now lack part of the normal cellular control; these therapeutic T cells could themselves become malignant. Transducing these modified T cells with a chimeric caspase-9-based safety switch as presented herein, would provide a safety switch that could avoid this result. Cells used in cellular therapy, that express a heterologous gene, such as a modified receptor, or a chimeric receptor, may be transduced with nucleic acid that encodes a chimeric caspase-9-based safety switch before, after, or at the same time, as the cells are transduced with the heterologous gene. Haploidentical stem cell transplantation While stem cell transplantation has proven an effective means of treating a wide variety of diseases involving hematopoietic stem cells and their progeny, a shortage of histocompatible donors has proved a major impediment to the widest application of the approach. The introduction of large panels of unrelated stem cell donors and or cord blood banks has helped to alleviate the problem, but many patients remain unsuited to either source. Even when a matched donor can be found, the elapsed time between commencing the search and collecting the stem cells usually exceeds three months, a delay that may doom many of the neediest patients. Hence there has been considerable interest in making use of HLA haploidentical family donors. Such donors may be parents, siblings or second-degree relatives. The problem of graft rejection may be overcome by a combination of appropriate conditioning and large doses of stem cells, while graft versus host disease (GvHD) may be prevented by extensive T cell-depletion of the donor graft. The immediate outcomes of such procedures have been gratifying, with engraftment rate > 90% and a severe GvHD rate of < 10% for both adults and children even in the absence of post transplant immunosuppression. Unfortunately the profound immunosuppression of the grafting procedure, coupled with the extensive T cell-depletion and HLA mismatching between donor and recipient lead to an extremely high rate of post-transplant infectious complications, and contributed to high incidence of disease relapse. Donor T cell infusion is an effective strategy for conferring anti-viral and anti-tumor immunity following allogeneic stem cell transplantation. Simple addback of T cells to the patients after haploidentical transplantation, however, cannot work; the frequency of alloreactive T cells is several orders of magnitude higher than the frequency of, for example, virus specific T lymphocytes. Methods are being developed to accelerate immune reconstitution by administrating donor T cells that have first been depleted of alloreactive ceils. One method of achieving this is stimulating donor T cells with recipient EBV-transformed B lymphoblastoid cell lines (LCLs). Alloreactive T cells upregulate CD25 expression, and are eliminated by a CD25 Mab immunotoxin conjugate, RFT5-SMPT-dgA. This compound consists of a murine lgG1 anti-CD25 (IL-2 receptor alpha chain) conjugated via a hetero-bifunctional crosslinker [N-succinimidyloxycarbonyl-alpha- methyl-d- (2-pyridylthio) toluene] to chemically deglycosylated ricin A chain (dgA). Treatment with CD25 immunotoxin after LCL stimulation depletes >90% of alloreactive cells. In a phase I clinical study, using CD25 immunotoxin to deplete alloreactive lymphocytes immune reconstitution after allodepleted donor T cells were infused at 2 dose levels into recipients of T-cell- depleted haploidentical SCT. Eight patients were treated at 10<com:Sup>4< / com:Sup> cells / kg / dose, and 8 patients received 10° cells / kg / dose. Patients receiving 10° cells / kg / dose showed significantly improved T- cell recovery at 3, 4, and 5 months after SCT compared with those receiving 10<com:Sup>4< / com:Sup> cells / kg / dose (P < .05). Accelerated T-cell recovery occurred as a result of expansion of the effector memory <com:Math><semantics>(CD45RA(−)CCR−7(−))<annotation encoding="application / x-tex">(CD45RA(-)CCR-7(-))< / annotation>< / semantics>< / com:Math> population <com:Math><semantics>(P<.05)<annotation encoding="application / x-tex">(P < .05)< / annotation>< / semantics>< / com:Math>, suggesting that protective T-cell responses are likely to be long lived. T-cell-receptor signal joint excision circles (TRECs) were not detected in reconstituting T cells in dose-level 2 patients, indicating they are likely to be derived from the infused allodepleted cells. Spectratyping of the T cells at 4 months demonstrated a polyclonal Vbeta repertoire. Using tetramer and enzyme-linked immunospot (ELISpot) assays, cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-specific responses in 4 of 6 evaluable patients at dose level 2 as early as 2 to 4 months after transplantation, whereas such responses were not observed until 6 to 12 months in dose-level 1 patients. The incidence of significant acute (2 of 16) and chronic graft-versus-host disease (GvHD; 2 of 15) was low. These data demonstrate that allodepleted donor T cells can be safely used to improve T-cell recovery after haploidentical SCT. The amount of cells infused was subsequently escalated to 10<com:Sup>6< / com:Sup> cells / kg without evidence of GvHD. Although this approach reconstituted antiviral immunity, relapse remained a major problem and 6 patients transplanted for high risk leukemia relapsed and died of disease. Higher T cell doses are therefore useful to reconstitute anti-tumor immunity and to provide the hoped-for anti-tumor effect, since the estimated frequency of tumor-reactive precursors is 1 to 2 logs less than frequency of viral-reactive precursors. However, in some patients, these doses of cells will be sufficient to trigger GvHD even after allodepletion (Hurley CK, et al., Biol Blood Marrow Transplant 2003;9:610- 615; Dey BR, et al., Br.J Haematol. 2006;135:423-437; Aversa F, et al., N Engl J Med 1998;339:1186-1193; Aversa F, et al., J C lin.On col. 2005;23:3447-3454; Lang P, Mol.Dis. 2004;33:281-287; Kolb HJ, et al., Blood 2004;103:767-776; Gottschalk S, et al., Annu.Rev.Med 2005;56:29-44; Bleakley M, et al., Nat.Rev.Cancer 2004;4:371-380; Andre-Schmutz I, et al., Lancet 2002;360:130-137; Solomon SR, et al., Blood 2005;106:1123-1129; Amrolia PJ, et al., Blood 2006;108:1797-1808; Amrolia PJ, et al., Blood 2003; Ghetie V, et al., J Immunol Methods 1991;142:223-230; Molldrem JJ, et al., Cancer Res 1999;59:2675-2681; Rezvani K, et al., Clin.Cancer Res. 2005;1 1:8799-8807; Rezvani K, et al., Blood 2003;102:2892-2900). Graft versus Host Disease (GvHD) Graft versus Host Disease is a condition that sometimes occurs after the transplantation of donor immunocompetent cells, for example, T cells, into a recipient. The transplanted cells recognize the recipient's cells as foreign, and attack and destroy them. This condition can be a dangerous effect of T cell transplantation, especially when associated with haploidentical stem cell transplantation. Sufficient T cells should be infused to provide the beneficial effects, such as, for example, the reconstitution of an immune system and the graft anti-tumor effect. But, the number of T cells that can be transplanted can be limited by the concern that the transplant will result in severe graft versus host disease. Graft versus Host Disease may be staged as indicated in the following tables: Table 1 Staging [Image disponible dans le document PDF, Image available in the PDF document] Acute GvHD grading may be performed by the consensus conference criteria (Przepiorka D et al., 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant 1995;15:825- 828). Grading Index of Acute GvHD [Image disponible dans le document PDF, Image available in the PDF document] Inducible caspase-9 as a "Safety Switch" for Cell Therapy and for Genetically Engineered Cell Transplantation By reducing the effect of graft versus host disease is meant, for example, a decrease in the GvHD symptoms so that the patient may be assigned a lower level stage, or, for example, a reduction of a symptom of graft versus host disease by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. A reduction in the effect of graft versus host disease may also be measured by detection of a reduction in activated T cells involved in the GvHD reaction, such as, for example, a reduction of cells that express the marker protein, for example CD19, and express CD3 (CD3+ CD19<com:Sup>+< / com:Sup> cells, for example) by at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. Provided herein is an alternative suicide gene strategy that is based on human proapoptotic molecules fused with an FKBP variant that is optimized to bind a chemical inducer of dimerization (CID) (Clackson T, et al., Proc Natl Acad Sci U S A. 1998, 95:10437-10442), m AP1903, a synthetic drug that has proven safe in healthy volunteers (Iuliucci JD, et al., J Clin Pharmacol. 2001, 41:870-879). Administration of this small molecule results in cross-linking and activation of the proapoptotic target molecules. The application of this inducible system in human T lymphocytes has been explored using Fas or the death effector domain (DED) of the Fas- associated death domain-containing protein (FADD) as proapoptotic molecules. Up to 90% of T cells transduced with these inducible death molecules underwent apoptosis after administration of CID (Thomis DC, et al., Blood. 2001, 97:1249-1257; Spencer DM, et al., Curr Biol. 1996, 6: 839- 847; Fan L, et al., Hum Gene Ther. 1999, 10: 2273-2285; Berger C, et al., Blood. 2004, 103:1261- 1269; Junker K, et al., Gene Ther. 2003, 10:1189-197). This suicide gene strategy may be used in any appropriate cell used for cell therapy including, for example, hematopoietic stem cells, and other progenitor cells, including, for example, mesenchymal stromal cells, embryonic stem cells, and inducible pluripotent stem cells. Therefore, this safety switch, catalyzed by caspase-9, may be used where there is a condition in the cell therapy patient that requires the removal of the transfected or transduced therapeutic cells. Therapeutic cells include, for example, any cell used for therapeutic treatment of a disease or condition, and include, for example, therapeutic cells selected from the group consisting of hematopoietic stem cells, inducible progenitor cells (iPS), embryonic stem (ES) cells, mesenchymal stem cells, plasma (B) cells, myocytes and T cells. Conditions where the cells may need to be removed include, for example, GvHD, inappropriate differentiation of the cells into more mature cells of the wrong tissue or cell type, and other toxicities. To activate the caspase-9 switch in the case of inappropriate differentiation, it is possible to use tissue specific promoters. For example, where a progenitor cell differentiates into bone and fat cells, and the fat cells are not desired, the vector used to transfect or transduce the progenitor cell may have a fat cell specific promoter that is operably linked to the caspase-9 nucleotide sequence. In this way, should the cells differentiate into fat cells, upon administration of the multimer ligand, apoptosis of the inappropriately differentiated fat cells should result. The methods may be used, for example, for any disorder that can be alleviated by cell therapy, including cancer, cancer in the blood or bone marrow, other blood or bone marrow borne diseases such as sickle cell anemia and metachromic leukodystrophy, and any disorder that can be alleviated by a stem cell transplantation, for example blood or bone marrow disorders such as sickle cell anemia or metachromal leukodystrophy. The efficacy of adoptive immunotherapy may be enhanced by rendering the therapeutic T cells resistant to immune evasion strategies employed by tumor cells. In vitro studies have shown that this can be achieved by transduction with a dominant-negative receptor or an immunomodulatory cytokine (Bollard CM, et al., Blood. 2002, 99:3179-3187: Wagner HJ, et al., Cancer Gene Ther. 2004, 11:81-91). Moreover, transfer of antigen-specific T-cell receptors allows for the application of T-cell therapy to a broader range of tumors (Pule M, et al., Cytotherapy, 2003, 5:211-226; Schumacher TN, Nat Rev Immunol. 2002, 2:512-519). A suicide system for engineered human T cells was developed and tested to allow their subsequent use in clinical studies. caspase-9 has been modified and shown to be stably expressed in human T lymphocytes without compromising their functional and phenotypic characteristics while demonstrating sensitivity to CID, even in T cells that have upregulated antiapoptotic molecules. (Straathof, K.C., et al., 2005, Blood 105:4248- 54). In genetically modified cells used for gene therapy, the gene may be a heterologous polynucleotide sequence derived from a source other than the cell that is being used to express the gene. The gene is derived from a prokaryotic or eukaryotic source such as a bacterium, a virus, yeast, a parasite, a plant, or even an animal. The heterologous DNA also is derived from more than one source, i.e., a multigene construct or a fusion protein. The heterologous DNA also may include a regulatory sequence, which is derived from one source and the gene from a different source. Or, the heterologous DNA may include regulatory sequences that are used to change the normal expression of a cellular endogenous gene. Other Caspase molecules Caspase polypeptides other than caspase-9 that may be encoded by the chimeric polypeotides of the current technology include, for example, Caspase-1, Caspase-3, and Caspase-8. Discussions of these Caspase polypeptides may be found in, for example, MacCorkle, R.A., et al., Proc. Natl. Acad. Sci. U.S.A. (1998) 95:3655-3660; and Fan, L., et al. (1999) Human Gene Therapy 10:2273- 2285). Engineering Expression Constructs Expression constructs encode a multimeric ligand binding region and a caspase-9 polypeptide, or, in certain embodiments a multimeric ligand binding region and a caspase-9 polypeptide linked to a marker polypeptide, all operatively linked. For purposes of this discussion, and for general references to the caspase-9 polypeptide, the term "caspase-9 polypeptide" is meant to include general references to modified caspase-9 polypeptides. In general, the term "operably linked" is meant to indicate that the promoter sequence is functionally linked to a second sequence, wherein, for example, the promoter sequence initiates and mediates transcription of the DNA corresponding to the second sequence. The caspase-9 polypeptide may be full length or truncated. In certain embodiments, the marker polypeptide is linked to the caspase-9 polypeptide. For example, the marker polypeptide may be linked to the caspase-9 polypeptide via a polypeptide sequence, such as, for example, a cleavable 2A-like sequence. The marker polypeptide may be, for example, CD19, <com:Math><semantics>Δ<annotation encoding="application / x-tex">\Delta< / annotation>< / semantics>< / com:Math>CD19, or may be, for example, a heterologous protein, selected to not affect the activity of the chimeric caspase polypeptide. In some embodiments, the polynucleotide may encode the caspase-9 polypeptide and a heterologous protein, which may be, for example a marker polypeptide and may be, for example, a chimeric antigen receptor. The heterologous polypeptide, for example, the chimeric antigen receptor, may be linked to the caspase-9 polypeptide via a polypeptide sequence, such as, for example, a cleavable 2A-like sequence. 2A-like sequences, or "cleavable" 2A sequences, are derived from, for example, many different viruses, including, for example, from Thosea asigna. These sequences are sometimes also known as "peptide skipping sequences." When this type of sequence is placed within a cistron, between two peptides that are intended to be separated, the ribosome appears to skip a peptide bond, in the case of Thosea asigna sequence, the bond between the Gly and Pro amino acids is omitted. This leaves two polypeptides, in this case the caspase-9 polypeptide and the marker polypeptide. When this sequence is used, the peptide that is encoded 5' of the 2A sequence may end up with additional amino acids at the carboxy terminus, including the Gly residue and any upstream in the 2A sequence. The peptide that is encoded 3' of the 2A sequence may end up with additional amino acids at the amino terminus, including the Pro residue and any downstream in the 2A sequence. "2A" or "2A-like" sequences are part of a large family of peptides that can cause peptide bond-skipping. Various 2A sequences have been characterized (e.g., F2A, P2A, T2A), and are examples of 2A-like sequences that may be used in the polypeptides of the present application... The expression construct may be inserted into a vector, for example a viral vector or plasmid. The steps of the methods provided may be performed using any suitable method, these methods include, without limitation, methods of transducing, transforming, or otherwise providing nucleic acid to the antigen-presenting cell, presented herein. In some embodiments, the truncated caspase-9 polypeptide is encoded by the nucleotide sequence of SEQ ID NO 8, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, or a functionally equivalent fragment thereof, with or without DNA linkers, or has the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28 or a functionally equivalent fragment thereof. In some embodiments, the CD19 polypeptide is encoded by the nucleotide sequence of SEQ ID NO 14, or a functionally equivalent fragment thereof, with or without DNA linkers, or has the amino acid sequence of SEQ ID NO: 15, or a functionally equivalent fragment thereof. A functionally equivalent fragment of the caspase-9 polypeptide has substantially the same ability to induce apoptosis as the polypeptide of SEQ ID NO: 9, with at least 50%, 60%, 70%, 80%, 90%, or 95% of the activity of the polypeptide of SEQ ID NO: 9. A functionally equivalent fragment of the CD19 polypeptide has substantially the same ability as the polypeptide of SEQ ID No: 15, to act as a marker to be used to identify and select transduced or transfected cells, with at least 50%, 60%, 70%, 80%, 90%, or 95% of the marker polypeptide being detected when compared to the polypeptide of SEQ ID NO: 15, using standard detection techniques. More particularly, more than one ligand-binding domain or multimerization region may be used in the expression construct. Yet further, the expression construct contains a membrane-targeting sequence. Appropriate expression constructs may include a co-stimulatory polypeptide element on either side of the above FKBP ligand-binding elements. Ligand-binding Regions The ligand-binding ("dimerization") domain, or multimerization region, of the expression construct can be any convenient domain that will allow for induction using a natural or unnatural ligand, for example, an unnatural synthetic ligand. The multimerization region can be internal or external to the cellular membrane, depending upon the nature of the construct and the choice of ligand. A wide variety of ligand-binding proteins, including receptors, are known, including ligand-binding proteins associated with the cytoplasmic regions indicated above. As used herein the term "ligand- binding domain" can be interchangeable with the term "receptor". Of particular interest are ligand- binding proteins for which ligands (for example, small organic ligands) are known or may be readily produced. These ligand-binding domains or receptors include the FKBPs and cyclophilin receptors, the steroid receptors, the tetracycline receptor, the other receptors indicated above, and the like, as well as "unnatural" receptors, which can be obtained from antibodies, particularly the heavy or light chain subunit, mutated sequences thereof, random amino acid sequences obtained by stochastic procedures, combinatorial syntheses, and the like. In certain embodiments, the ligand- binding region is selected from the group consisting of FKBP ligand-binding region, cyclophilin receptor ligand-binding region, steroid receptor ligand-binding region, cyclophilin receptors ligand- binding region, and tetracycline receptor ligand-binding region. Often, the ligand-binding region comprises a <com:Math><semantics>Fv′Fv|s<annotation encoding="application / x-tex">F_{v'}F_{v|s}< / annotation>< / semantics>< / com:Math> sequence. Sometimes, the <com:Math><semantics>Fv′Fv|s<annotation encoding="application / x-tex">F_{v'}F_{v|s}< / annotation>< / semantics>< / com:Math> sequence further comprises an additional <com:Math><semantics>Fv′<annotation encoding="application / x-tex">F_{v'}< / annotation>< / semantics>< / com:Math> sequence. Examples include, for example, those discussed in Kopytek, S.J., et al., Chemistry & Biology 7:313-321 (2000) and in Gestwicki, J.E., et al., Combinatorial Chem. & High Throughput Screening 10:667-675 (2007); Clackson T (2006) Chem Biol Drug Des 67:440-2; Clackson, T., in Chemical Biology: From Small Molecules to Systems Biology and Drug Design (Schreiber, s., et al., eds., Wiley, 2007)). For the most part, the ligand-binding domains or receptor domains will be at least about 50 amino acids, and fewer than about 350 amino acids, usually fewer than 200 amino acids, either as the natural domain or truncated active portion thereof. The binding domain may, for example, be small (<25 kDa, to allow efficient transfection in viral vectors), monomeric, nonimmunogenic, have synthetically accessible, cell permeable, nontoxic ligands that can be configured for dimerization. The receptor domain can be intracellular or extracellular depending upon the design of the expression construct and the availability of an appropriate ligand. For hydrophobic ligands, the binding domain can be on either side of the membrane, but for hydrophilic ligands, particularly protein ligands, the binding domain will usually be external to the cell membrane, unless there is a transport system for internalizing the ligand in a form in which it is available for binding. For an intracellular receptor, the construct can encode a signal peptide and transmembrane domain 5' or 3' of the receptor domain sequence or may have a lipid attachment signal sequence 5' of the receptor domain sequence. Where the receptor domain is between the signal peptide and the transmembrane domain, the receptor domain will be extracellular. The portion of the expression construct encoding the receptor can be subjected to mutagenesis for a variety of reasons. The mutagenized protein can provide for higher binding affinity, allow for discrimination by the ligand of the naturally occurring receptor and the mutagenized receptor, provide opportunities to design a receptor-ligand pair, or the like. The change in the receptor can involve changes in amino acids known to be at the binding site, random mutagenesis using combinatorial techniques, where the codons for the amino acids associated with the binding site or other amino acids associated with conformational changes can be subject to mutagenesis by changing the codon(s) for the particular amino acid, either with known changes or randomly, expressing the resulting proteins in an appropriate prokaryotic host and then screening the resulting proteins for binding. Antibodies and antibody subunits, e.g., heavy or light chain, particularly fragments, more particularly all or part of the variable region, or fusions of heavy and light chain to create high- affinity binding, can be used as the binding domain. Antibodies that are contemplated include ones that are an ectopically expressed human product, such as an extracellular domain that would not trigger an immune response and generally not expressed in the periphery (i.e., outside the CNS / brain area). Such examples, include, but are not limited to low affinity nerve growth factor receptor (LNGFR), and embryonic surface proteins (i.e., carcinoembryonic antigen). Yet further, antibodies can be prepared against haptenic molecules, which are physiologically acceptable, and the individual antibody subunits screened for binding affinity. The cDNA encoding the subunits can be isolated and modified by deletion of the constant region, portions of the variable region, mutagenesis of the variable region, or the like, to obtain a binding protein domain that has the appropriate affinity for the ligand. In this way, almost any physiologically acceptable haptenic compound can be employed as the ligand or to provide an epitope for the ligand. Instead of antibody units, natural receptors can be employed, where the binding domain is known and there is a useful ligand for binding. Oligomerization | The transduced signal will normally result from ligand-mediated oligomerization of the chimeric protein molecules, i.e., as a result of oligomerization following ligand-binding, although other binding events, for example allosteric activation, can be employed to initiate a signal. The construct of the chimeric protein will vary as to the order of the various domains and the number of repeats of an individual domain. For multimerizing the receptor, the ligand for the ligand-binding domains / receptor domains of the chimeric surface membrane proteins will usually be multimeric in the sense that it will have at least two binding sites, with each of the binding sites capable of binding to the ligand receptor domain. By "multimeric ligand binding region" is meant a ligand binding region that binds to a multimeric ligand. The term "multimeric ligands" include dimeric ligands. A dimeric ligand will have two binding sites capable of binding to the ligand receptor domain. Desirably, the subject ligands will be a dimer or higher order oligomer, usually not greater than about tetrameric, of small synthetic organic molecules, the individual molecules typically being at least about 150 Da and less than about 5 kDa, usually less than about 3 kDa. A variety of pairs of synthetic ligands and receptors can be employed. For example, in embodiments involving natural receptors, dimeric FK506 can be used with an FKBP12 receptor, dimerized cyclosporin A can be used with the cyclophilin receptor, dimerized estrogen with an estrogen receptor, dimerized glucocorticoids with a glucocorticoid receptor, dimerized tetracycline with the tetracycline receptor, dimerized vitamin D with the vitamin D receptor, and the like. Alternatively higher orders of the ligands, e.g., trimeric can be used. For embodiments involving unnatural receptors, e.g., antibody subunits, modified antibody subunits, single chain antibodies comprised of heavy and light chain variable regions in tandem, separated by a flexible linker domain, or modified receptors, and mutated sequences thereof, and the like, any of a large variety of compounds can be used. A significant characteristic of these ligand units is that each binding site is able to bind the receptor with high affinity and they are able to be dimerized chemically. Also, methods are available to balance the hydrophobicity / hydrophilicity of the ligands so that they are able to dissolve in serum at functional levels, yet diffuse across plasma membranes for most applications. In certain embodiments, the present methods utilize the technique of chemically induced dimerization (CID) to produce a conditionally controlled protein or polypeptide. In addition to this technique being inducible, it also is reversible, due to the degradation of the labile dimerizing agent or administration of a monomeric competitive inhibitor. The CID system uses synthetic bivalent ligands to rapidly crosslink signaling molecules that are fused to ligand-binding domains. This system has been used to trigger the oligomerization and activation of cell surface (Spencer, D. M., et al., Science, 1993. 262: p. 1019-1024; Spencer D. M. et al., Curr Biol 1996, 6:839-847; Blau, C. A. et al., Proc Natl Acad.Sci. USA 1997, 94:3076-3081), or cytosolic proteins (Luo, Z. et al., Nature 1996,383:181-185; MacCorkle, R. A. et al., Proc Natl Acad Sci USA 1998, 95:3655-3660), the recruitment of transcription factors to DNA elements to modulate transcription (Ho, S. N. et al., Nature 1996, 382:822-826; Rivera, V. M. et al., Nat.Med. 1996, 2:1028-1032) or the recruitment of signaling molecules to the plasma membrane to stimulate signaling (Spencer D. M. et al., Proc.Natl.Acad.Sci. USA 1995, 92:9805-9809; Holsinger, L. J. et al., Proc.Natl.Acad.Sci. USA 1995, 95:9810-9814). The CID system is based upon the notion that surface receptor aggregation effectively activates downstream signaling cascades. In the simplest embodiment, the CID system uses a dimeric analog of the lipid permeable immunosuppressant drug, FK506, which loses its normal bioactivity while gaining the ability to crosslink molecules genetically fused to the FK506-binding protein, FKBP12. By fusing one or more FKBPs to caspase-9, one can stimulate caspase-9 activity in a dimerizer drug-dependent, but ligand and ectodomain-independent manner. This provides the system with temporal control, reversibility using monomeric drug analogs, and enhanced specificity. The high affinity of third-generation AP20187 / AP1903 CIDs for their binding domain, FKBP12, permits specific activation of the recombinant receptor in vivo without the induction of non-specific side effects through endogenous FKBP12. FKBP12 variants having amino acid substitutions and deletions, such as FKBP12v36, that bind to a dimerizer drug, may also be used. In addition, the synthetic ligands are resistant to protease degradation, making them more efficient at activating receptors in vivo than most delivered protein agents. The ligands used are capable of binding to two or more of the ligand-binding domains. The chimeric proteins may be able to bind to more than one ligand when they contain more than one ligand-binding domain. The ligand is typically a non-protein or a chemical. Exemplary ligands include, but are not limited to FK506 (e.g., FK1012). Other ligand binding regions may be, for example, dimeric regions, or modified ligand binding regions with a wobble substitution, such as, for example, FKBP12(V36): The human 12 kDa FK506-binding protein with an F36 to V substitution, the complete mature coding sequence (amino acids 1-107), provides a binding site for synthetic dimerizer drug AP1903 (Jemal, A. et al., CA Cancer J. Clinic. 58, 71-96 (2008); Scher, H.I. and Kelly, W.K., Journal of Clinical Oncology 11, 1566-72 (1993)). Two tandem copies of the protein may also be used in the construct so that higher-order oligomers are induced upon cross-linking by AP1903. F36V'-FKBP: F36V'-FKBP is a codon—wobbled version of F36V-FKBP. It encodes the identical polypeptide sequence as F36V-FKPB but has only 62% homology at the nucleotide level. F36V'-FKBP was designed to reduce recombination in retroviral vectors (Schellhammer, P.F. et al., J. Urol. 157, 1731-5 (1997)). F36V'-FKBP was constructed by a PCR assembly procedure. The transgene contains one copy of F36V'-FKBP linked directly to one copy of F36V- FKBP. In some embodiments, the ligand is a small molecule. The appropriate ligand for the selected ligand-binding region may be selected. Often, the ligand is dimeric, sometimes the ligand is a dimeric FK506 or a dimeric FK506-like analog. In certain embodiments, the ligand is AP1903 (CAS Index Name: 2-Piperidinecarboxylic acid, 1-[(2S)-1-oxo-2-(3,4,5-trimethoxyphenyl)butyl]-, 1,2-ethanediylbis[imino(2-oxo-2,1-ethanediyl)oxy-3,1-phenylene[(1R)-3-(3,4- dimethoxyphenyl)propylidene]] ester, [2S-[1(R*),2R*[S*[1(R*),2R*]]]]]-(9CI) CAS Registry Number: 195514-63-7; Molecular Formula: C78H98N4O20 Molecular Weight: 1411.65). In certain embodiments, the ligand is AP20187. In certain embodiments, the ligand is an AP20187 analog, such as, for example, AP1510. In some embodiments, certain analogs will be appropriate for the FKBP12, and certain analogs appropriate for the wobbled version of FKBP12. In certain embodiments, one ligand binding region is included in the chimeric protein. In other embodiments, two or more ligand binding regions are included. Where, for example, the ligand binding region is FKBP12, where two of these regions are included, one may, for example, be the wobbled version. Other dimerization systems contemplated include the coumermycin / DNA gyrase B system. Coumermycin-induced dimerization activates a modified Raf protein and stimulates the MAP kinase cascade. See Farrar et al., 1996. AP1903 for Injection AP1903 is manufactured by Alphora Research Inc. and AP1903 Drug Product for Injection is made by Formatech Inc. It is formulated as a 5 mg / mL solution of AP1903 in a 25% solution of the non- ionic solubilizer Solutol HS 15 (250 mg / mL, BASF). At room temperature, this formulation is a clear, slightly yellow solution. Upon refrigeration, this formulation undergoes a reversible phase transition, resulting in a milky solution. This phase transition is reversed upon re-warming to room temperature. The fill is 2.33 mL in a 3 mL glass vial (~10 mg AP1903 for Injection total per vial). AP1903 is removed from the refrigerator the night before the patient is dosed and stored at a temperature of approximately 21 °C overnight, so that the solution is clear prior to dilution. The solution is prepared within 30 minutes of the start of the infusion in glass or polyethylene bottles or non-DEHP bags and stored at approximately 21 °C prior to dosing. All study medication is maintained at a temperature between 2 degrees C and 8 degrees C, protected from excessive light and heat, and stored in a locked area with restricted access. Upon determining a need to administer AP1903 and induce the inducible caspase-9 polypeptide, patients may be, for example, administered a single fixed dose of AP1903 for Injection (0.4 mg / kg) via IV infusion over 2 hours, using a non-DEHP, non-ethylene oxide sterilized infusion set. The dose of AP1903 is calculated individually for all patients, and is not be recalculated unless body weight fluctuates by ≥10%. The calculated dose is diluted in 100 mL in 0.9% normal saline before infusion. In a previous Phase I study of AP1903, 24 healthy volunteers were treated with single doses of AP1903 for Injection at dose levels of 0.01, 0.05, 0.1, 0.5 and 1.0 mg / kg infused IV over 2 hours. AP1903 plasma levels were directly proportional to dose, with mean C<com:Sub>max< / com:Sub> values ranging from approximately 10 - 1275 ng / mL over the 0.01 - 1.0 mg / kg dose range. Following the initial infusion period, blood concentrations demonstrated a rapid distribution phase, with plasma levels reduced to approximately 18, 7, and 1% of maximal concentration at 0.5, 2 and 10 hours post- dose, respectively. AP1903 for Injection was shown to be safe and well tolerated at all dose levels and demonstrated a favorable pharmacokinetic profile. Iuliucci JD, et al., J Clin Pharmacol. 41: 870-9, 2001. The fixed dose of AP1903 for injection used, for example, may be 0.4 mg / kg intravenously infused over 2 hours. The amount of AP1903 needed in vitro for effective signaling of cells is 10 – 100 nM (1600 Da MW). This equates to <com:Math><semantics>16−160μg / L<annotation encoding="application / x-tex">16 - 160 \mu g / L< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>∼0.016−1.6mg / kg<annotation encoding="application / x-tex">\sim 0.016 - 1.6 m g / kg< / annotation>< / semantics>< / com:Math> (1.6 - 160 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>). Doses up to 1 mg / kg were well-tolerated in the Phase I study of AP1903 described above. Selectable Markers In certain embodiments, the expression constructs contain nucleic acid constructs whose expression is identified in vitro or in vivo by including a marker in the expression construct. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct. Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants. For example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. Alternatively, enzymes such as Herpes Simplex Virus-I thymidine kinase (tk) are employed. Immunologic surface markers containing the extracellular, non-signaling domains or various proteins (e.g. CD34, CD19, LNGFR) also can be employed, permitting a straightforward method for magnetic or fluorescence antibody-mediated sorting. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers include, for example, reporters such as GFP, EGFP, beta-gal or chloramphenicol acetyltransferase (CAT). In certain embodiments, the marker protein, such as, for example, CD19 is used for selection of the cells for transfusion, such as, for example, in immunomagnetic selection. Control Regions Promoters The particular promoter employed to control the expression of a polynucleotide sequence of interest is not believed to be important, so long as it is capable of directing the expression of the polynucleotide in the targeted cell. Thus, where a human cell is targeted the polynucleotide sequence-coding region may, for example, be placed adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter. In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, B-actin, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Selection of a promoter that is regulated in response to specific physiologic or synthetic signals can permit inducible expression of the gene product. For example in the case where expression of a transgene, or transgenes when a multicistronic vector is utilized, is toxic to the cells in which the vector is produced in, it is desirable to prohibit or reduce expression of one or more of the transgenes. Examples of transgenes that are toxic to the producer cell line are pro-apoptotic and cytokine genes. Several inducible promoter systems are available for production of viral vectors where the transgene products are toxic (add in more inducible promoters). The ecdysone system (Invitrogen, Carlsbad, CA) is one such system. This system is designed to allow regulated expression of a gene of interest in mammalian cells. It consists of a tightly regulated expression mechanism that allows virtually no basal level expression of the transgene, but over 200-fold inducibility. The system is based on the heterodimeric ecdysone receptor of Drosophila, and when ecdysone or an analog such as muristerone A binds to the receptor, the receptor activates a promoter to turn on expression of the downstream transgene high levels of mRNA transcripts are attained. In this system, both monomers of the heterodimeric receptor are constitutively expressed from one vector, whereas the ecdysone-responsive promoter, which drives expression of the gene of interest, is on another plasmid. Engineering of this type of system into the gene transfer vector of interest would therefore be useful. Cotransfection of plasmids containing the gene of interest and the receptor monomers in the producer cell line would then allow for the production of the gene transfer vector without expression of a potentially toxic transgene. At the appropriate time, expression of the transgene could be activated with ecdysone or muristeron A. Another inducible system that may be useful is the Tet-Off<com:Sup>TM< / com:Sup> or Tet-On<com:Sup>TM< / com:Sup> system (Clontech, Palo Alto, CA) originally developed by Gossen and Bujard (Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89:5547-5551, 1992; Gossen et al., Science, 268:1766-1769, 1995). This system also allows high levels of gene expression to be regulated in response to tetracycline or tetracycline derivatives such as doxycycline. In the Tet-On<com:Sup>TM< / com:Sup> system, gene expression is turned on in the presence of doxycycline, whereas in the Tet-Off<com:Sup>TM< / com:Sup> system, gene expression is turned on in the absence of doxycycline. These systems are based on two regulatory elements derived from the tetracycline resistance operon of E. coli, the tetracycline operator sequence to which the tetracycline repressor binds, and the tetracycline repressor protein. The gene of interest is cloned into a plasmid behind a promoter that has tetracycline-responsive elements present in it. A second plasmid contains a regulatory element called the tetracycline-controlled transactivator, which is composed, in the Tet-Off<com:Sup>TM< / com:Sup> system, of the VP16 domain from the herpes simplex virus and the wild-type tertracycline repressor. Thus in the absence of doxycycline, transcription is constitutively on. In the Tet-On™ system, the tetracycline repressor is not wildtype and in the presence of doxycycline activates transcription. For gene therapy vector production, the Tet-Off™ system may be used so that the producer cells could be grown in the presence of tetracycline or doxycycline and prevent expression of a potentially toxic transgene, but when the vector is introduced to the patient, the gene expression would be constitutively on. In some circumstances, it is desirable to regulate expression of a transgene in a gene therapy vector. For example, different viral promoters with varying strengths of activity are utilized depending on the level of expression desired. In mammalian cells, the CMV immediate early promoter is often used to provide strong transcriptional activation. The CMV promoter is reviewed in Donnelly, J.J., et al., 1997. Annu. Rev. Immunol. 15:617-48. Modified versions of the CMV promoter that are less potent have also been used when reduced levels of expression of the transgene are desired. When expression of a transgene in hematopoietic cells is desired, retroviral promoters such as the LTRs from MLV or MMTV are often used. Other viral promoters that are used depending on the desired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters such as from the E1A, E2A, or MLP region, AAV LTR, HSV-TK, and avian sarcoma virus. In other examples, promoters may be selected that are developmentally regulated and are active in particular differentiated cells. Thus, for example, a promoter may not be active in a pluripotent stem cell, but, for example, where the pluripotent stem cell differentiates into a more mature cell, the promoter may then be activated. Similarly tissue specific promoters are used to effect transcription in specific tissues or cells so as to reduce potential toxicity or undesirable effects to non-targeted tissues. These promoters may result in reduced expression compared to a stronger promoter such as the CMV promoter, but may also result in more limited expression, and immunogenicity (Bojak, A., et al., 2002. Vaccine. 20:1975-79; Cazeaux., N., et al., 2002. Vaccine 20:3322-31). For example, tissue specific promoters such as the PSA associated promoter or prostate-specific glandular kallikrein, or the muscle creatine kinase gene may be used where appropriate. Examples of tissue specific or differentiation specific promoters include, but are not limited to, the following: B29 (B cells); CD14 (monocytic cells); CD43 (leukocytes and platelets); CD45 (hematopoietic cells); CD68 (macrophages); desmin (muscle); elastase-1 (pancreatic acinar cells); endoglin (endothelial cells); fibronectin (differentiating cells, healing tissues); and Flt-1 (endothelial cells); GFAP (astrocytes). In certain indications, it is desirable to activate transcription at specific times after administration of the gene therapy vector. This is done with such promoters as those that are hormone or cytokine regulatable. Cytokine and inflammatory protein responsive promoters that can be used include K and T kininogen (Kageyama et al., (1987) J. Biol. Chem., 262,2345-2351), c-fos, TNF-alpha, C- reactive protein (Arcone, et al., (1988) Nucl. Acids Res., 16(8), 3195-3207), haptoglobin (Oliviero et al., (1987) EMBO J., 6, 1905-1912), serum amyloid A2, C / EBP alpha, IL-1, IL-6 (Poli and Cortese, (1989) Proc. Nat'l Acad. Sci. USA, 86,8202-8206), Complement C3 (Wilson et al., (1990) Mol. Cell. Biol., 6181-6191), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, (1988) Mol Cell Biol, 8,42-51), alpha-1 antitrypsin, lipoprotein lipase (Zechner et al., Mol. Cell. Biol., 2394-2401, 1988), angiotensinogen (Ron, et al., (1991) Mol. Cell. Biol., 2887-2895), fibrinogen, c-jun (inducible by phorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide), collagenase (induced by phorbol esters and retinoic acid), metallothionein (heavy metal and glucocorticoid inducible), Stromelysin (inducible by phorbol ester, interleukin-1 and EGF), alpha-2 macroglobulin and alpha-1 anti-chymotrypsin. Other promoters include, for example, SV40, MMTV, Human Immunodeficiency Virus (MV), Moloney virus, ALV, Epstein Barr virus, Rous Sarcoma virus, human actin, myosin, hemoglobin, and creatine. It is envisioned that any of the above promoters alone or in combination with another can be useful depending on the action desired. Promoters, and other regulatory elements, are selected such that they are functional in the desired cells or tissue. In addition, this list of promoters should not be construed to be exhaustive or limiting; other promoters that are used in conjunction with the promoters and methods disclosed herein. Enhancers Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Early examples include the enhancers associated with immunoglobulin and T cell receptors that both flank the coding sequence and occur within several introns. Many viral promoters, such as CMV, SV40, and retroviral LTRs are closely associated with enhancer activity and are often treated like single elements. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole stimulates transcription at a distance and often independent of orientation; this need not be true of a promoter region or its component elements. On the other hand, a promoter has one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization. A subset of enhancers is locus-control regions (LCRs) that can not only increase transcriptional activity, but (along with insulator elements) can also help to insulate the transcriptional element from adjacent sequences when integrated into the genome. Any promoter / enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) can be used to drive expression of the gene, although many will restrict expression to a particular tissue type or subset of tissues (reviewed in, for example, Kutzler, M.A., and Weiner, D.B., 2008. Nature Reviews Genetics 9:776-88). Examples include, but are not limited to, enhancers from the human actin, myosin, hemoglobin, muscle creatine kinase, sequences, and from viruses CMV, RSV, and EBV. Appropriate enhancers may be selected for particular applications. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct. Polyadenylation Signals Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the present methods, and any such sequence is employed such as human or bovine growth hormone and SV40 polyadenylation signals and LTR polyadenylation signals. One non-limiting example is the SV40 polyadenylation signal present in the pCEP3 plasmid (Invitrogen, Carlsbad, California). Also, contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences. Termination or poly(A) signal sequences may be, for example, positioned about 11-30 nucleotides downstream from a conserved sequence (AAUAAA) at the 3' end of the mRNA (Montgomery, D.L., et al., 1993. DNA Cell Biol. 12:777-83; Kutzler, M.A., and Weiner, D.B., 2008. Nature Rev. Gen. 9:776-88). Initiation Signals and Internal Ribosome Binding Sites A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. The initiation codon is placed in-frame with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements. In certain embodiments, the use of internal ribosome entry sites (IRES) elements is used to create multigene, or polycistronic messages. IRES elements are able to bypass the ribosome-scanning model of 5' methylated cap-dependent translation and begin translation at internal sites (Pelletier and Sonenberg, Nature, 334:320-325, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been discussed (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, Nature, 353:90-94, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see U.S. Patent Nos. 5,925,565 and 5,935,819). Sequence Optimization Protein production may also be increased by optimizing the codons in the transgene. Species specific codon changes may be used to increase protein production. Also, codons may be optimized to produce an optimized RNA, which may result in more efficient translation. By optimizing the codons to be incorporated in the RNA, elements such as those that result in a secondary structure that causes instability, secondary mRNA structures that can, for example, inhibit ribosomal binding, or cryptic sequences that can inhibit nuclear export of mRNA can be removed (Kutzler, M.A., and Weiner, D.B., 2008. Nature Rev. Gen. 9:776-88; Yan, J. et al., 2007. Mol. Ther. 15:411-21; Cheung, Y.K., et al., 2004. Vaccine 23:629-38; Narum, D.L., et al., 2001. 69:7250-55; Yadava, A., and Ockenhouse, C.F., 2003. Infect. Immun. 71:4962-69; Smith, J.M., et al., 2004. AIDS Res. Hum. Retroviruses 20:1335-47; Zhou, W., et al., 2002. Vet. Microbiol. 88:127- 51; Wu, X., et al., 2004. Biochem. Biophys. Res. Commun. 313:89-96; Zhang, W., et al., 2006. Biochem. Biophys. Res. Commun. 349:69-78; Deml, L.A., et al., 2001. J. Virol. 75:1099-11001; Schneider, R. M., et al., 1997. J. Virol. 71:4892-4903; Wang, S.D., et al., 2006. Vaccine 24:4531- 40; zur Megede, J., et al., 2000. J. Virol. 74:2628-2635). For example, the FBP12, the Caspase polypeptide, and the CD19 sequences may be optimized by changes in the codons. Leader Sequences Leader sequences may be added to enhance the stability of mRNA and result in more efficient translation. The leader sequence is usually involved in targeting the mRNA to the endoplasmic reticulum. Examples include the signal sequence for the HIV-1 envelope glycoprotein (Env), which delays its own cleavage, and the IgE gene leader sequence (Kutzler, M.A., and Weiner, D.B., 2008. Nature Rev. Gen. 9:776-88; Li, V., et al., 2000. Virology 272:417-28; Xu, Z.L., et al. 2001. Gene 272:149-56; Malin, A.S., et al., 2000. Microbes Infect. 2:1677-85; Kutzler, M.A., et al., 2005. J. Immunol. 175:112-125; Yang, J.S., et al., 2002. Emerg. Infect. Dis. 8:1379-84; Kumar, S., et al., 2006. DNA Cell Biol. 25:383-92; Wang, S., et al., 2006. Vaccine 24:4531-40). The lgE leader may be used to enhance insertion into the endoplasmic reticulum (Tepler, I, et al. (1989) J. Biol. Chem. 264:5912). Expression of the transgenes may be optimized and / or controlled by the selection of appropriate methods for optimizing expression. These methods include, for example, optimizing promoters, delivery methods, and gene sequences, (for example, as presented in Laddy, D.J., et al., 2008. PLoS.ONE 3 e2517; Kutzler, M.A., and Weiner, D.B., 2008. Nature Rev. Gen. 9:776-88). Nucleic Acids A "nucleic acid" as used herein generally refers to a molecule (one, two or more strands) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase. A nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., an adenine "A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g., an A, a G, an uracil "U" or a C). The term "nucleic acid" encompasses the terms "oligonucleotide" and "polynucleotide," each as a subgenus of the term "nucleic acid." Nucleic acids may be, be at least, be at most, or be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides, or any range derivable therein, in length. Nucleic acids herein provided may have regions of identity or complementarity to another nucleic acid. It is contemplated that the region of complementarity or identity can be at least 5 contiguous residues, though it is specifically contemplated that the region is, is at least, is at most, or is about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 contiguous nucleotides. As used herein, "hybridization", "hybridizes" or "capable of hybridizing" is understood to mean forming a double or triple stranded molecule or a molecule with partial double or triple stranded nature. The term "anneal" as used herein is synonymous with "hybridize." The term "hybridization", "hybridize(s)" or "capable of hybridizing" encompasses the terms "stringent condition(s)" or "high stringency" and the terms "low stringency" or "low stringency condition(s)." As used herein "stringent condition(s)" or "high stringency" are those conditions that allow hybridization between or within one or more nucleic acid strand(s) containing complementary sequence(s), but preclude hybridization of random sequences. Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand. Such conditions are known, and are often used for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA transcript or a nucleic acid segment thereof, and the like. Stringent conditions may comprise low salt and / or high temperature conditions, such as provided by about 0.02 M to about 0.5 M NaCl at temperatures of about 42 degrees C to about 70 degrees C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleobase content of the target sequence(s), the charge composition of the nucleic acid(s), and the presence or concentration of formamide, tetramethylammonium chloride or other solvent(s) in a hybridization mixture. It is understood that these ranges, compositions and conditions for hybridization are mentioned by way of non-limiting examples only, and that the desired stringency for a particular hybridization reaction is often determined empirically by comparison to one or more positive or negative controls. Depending on the application envisioned varying conditions of hybridization may be employed to achieve varying degrees of selectivity of a nucleic acid towards a target sequence. In a non-limiting example, identification or isolation of a related target nucleic acid that does not hybridize to a nucleic acid under stringent conditions may be achieved by hybridization at low temperature and / or high ionic strength. Such conditions are termed "low stringency" or "low stringency conditions," and non-limiting examples of low stringency include hybridization performed at about 0.15 M to about 0.9 M NaCl at a temperature range of about 20 degrees C. to about 50 degrees C. The low or high stringency conditions may be further modified to suit a particular application. Nucleic Acid Modification Any of the modifications discussed below may be applied to a nucleic acid. Examples of modifications include alterations to the RNA or DNA backbone, sugar or base, and various combinations thereof. Any suitable number of backbone linkages, sugars and / or bases in a nucleic acid can be modified (e.g., independently about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, up to 100%). An unmodified nucleoside is any one of the bases adenine, cytosine, guanine, thymine, or uracil joined to the 1' carbon of beta-D-ribo-furanose. A modified base is a nucleotide base other than adenine, guanine, cytosine and uracil at a 1' position. Non-limiting examples of modified bases include inosine, purine, pyridin-4-one, pyridin-2- one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5- halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6- methyluridine), propyne, and the like. Other non-limiting examples of modified bases include nitropyrrolyl (e.g., 3-nitropyrrolyl), nitroindolyl (e.g., 4-, 5-, 6-nitroindolyl), hypoxanthinyl, isoinosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4- methylbenzimidazole, 3-methyl isocarbostyrilyl, 5-methyl isocarbostyrilyl, 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5- trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl and the like. In some embodiments, for example, a nucleic acid may comprise modified nucleic acid molecules, with phosphate backbone modifications. Non-limiting examples of backbone modifications include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and / or alkylsilyl modifications. In certain instances, a ribose sugar moiety that naturally occurs in a nucleoside is replaced with a hexose sugar, polycyclic heteroalkyl ring, or cyclohexenyl group. In certain instances, the hexose sugar is an allose, altrose, glucose, mannose, gulose, idose, galactose, talose, or a derivative thereof. The hexose may be a D-hexose, glucose, or mannose. In certain instances, the polycyclic heteroalkyl group may be a bicyclic ring containing one oxygen atom in the ring. In certain instances, the polycyclic heteroalkyl group is a bicyclo[2.2.1]heptane, a bicyclo[3.2.1]octane, or a bicyclo[3.3.1]nonane. Nitropyrrolyl and nitroindolyl nucleobases are members of a class of compounds known as universal bases. Universal bases are those compounds that can replace any of the four naturally occurring bases without substantially affecting the melting behavior or activity of the oligonucleotide duplex. In contrast to the stabilizing, hydrogen-bonding interactions associated with naturally occurring nucleobases, oligonucleotide duplexes containing 3-nitropyrrolyl nucleobases may be stabilized solely by stacking interactions. The absence of significant hydrogen-bonding interactions with nitropyrrolyl nucleobases obviates the specificity for a specific complementary base. In addition, 4-, 5- and 6-nitroindolyl display very little specificity for the four natural bases. Procedures for the preparation of 1-(2'-O-methyl-.beta.-D-ribofuranosyl)-5-nitroindole are discussed in Gaubert, G.; Wengel, J. Tetrahedron Letters 2004, 45, 5629. Other universal bases include hypoxanthinyl, isoinosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, and structural derivatives thereof. Difluorotolyl is a non-natural nucleobase that functions as a universal base. Difluorotolyl is an isostere of the natural nucleobase thymine. But unlike thymine, difluorotolyl shows no appreciable selectivity for any of the natural bases. Other aromatic compounds that function as universal bases are 4-fluoro-6-methylbenzimidazole and 4-methylbenzimidazole. In addition, the relatively hydrophobic isocarbostyrilyl derivatives 3-methyl isocarbostyrilyl, 5-methyl isocarbostyrilyl, and 3- methyl-7-propynyl isocarbostyrilyl are universal bases which cause only slight destabilization of oligonucleotide duplexes compared to the oligonucleotide sequence containing only natural bases. Other non-natural nucleobases include 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9- methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7- azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivates thereof. For a more detailed discussion, including synthetic procedures, of difluorotolyl, 4-fluoro-6- methylbenzimidazole, 4-methylbenzimidazole, and other non-natural bases mentioned above, see: Schweitzer et al., J. Org. Chem., 59:7238-7242 (1994); In addition, chemical substituents, for example cross-linking agents, may be used to add further stability or irreversibility to the reaction. Non-limiting examples of cross-linking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N- maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl) dithio]propioimidate. A nucleotide analog may also include a "locked" nucleic acid. Certain compositions can be used to essentially "anchor" or "lock" an endogenous nucleic acid into a particular structure. Anchoring sequences serve to prevent disassociation of a nucleic acid complex, and thus not only can prevent copying but may also enable labeling, modification, and / or cloning of the endogenous sequence. The locked structure may regulate gene expression (i.e. inhibit or enhance transcription or replication), or can be used as a stable structure that can be used to label or otherwise modify the endogenous nucleic acid sequence, or can be used to isolate the endogenous sequence, i.e. for cloning. Nucleic acid molecules need not be limited to those molecules containing only RNA or DNA, but further encompass chemically-modified nucleotides and non-nucleotides. The percent of non- nucleotides or modified nucleotides may be from 1% to 100% (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95%). Nucleic Acid Preparation In some embodiments, a nucleic acid is provided for use as a control or standard in an assay, or therapeutic, for example. A nucleic acid may be made by any technique known in the art, such as for example, chemical synthesis, enzymatic production or biological production. Nucleic acids may be recovered or isolated from a biological sample. The nucleic acid may be recombinant or it may be natural or endogenous to the cell (produced from the cell's genome). It is contemplated that a biological sample may be treated in a way so as to enhance the recovery of small nucleic acid molecules. Generally, methods may involve lysing cells with a solution having guanidinium and a detergent. Nucleic acid synthesis may also be performed according to standard methods. Non-limiting examples of a synthetic nucleic acid (e.g., a synthetic oligonucleotide), include a nucleic acid made by in vitro chemical synthesis using phosphotriester, phosphite, or phosphoramidite chemistry and solid phase techniques or via deoxynucleoside H-phosphonate intermediates. Various different mechanisms of oligonucleotide synthesis have been disclosed elsewhere. Nucleic acids may be isolated using known techniques. In particular embodiments, methods for isolating small nucleic acid molecules, and / or isolating RNA molecules can be employed. Chromatography is a process used to separate or isolate nucleic acids from protein or from other nucleic acids. Such methods can involve electrophoresis with a gel matrix, filter columns, alcohol precipitation, and / or other chromatography. If a nucleic acid from cells is to be used or evaluated, methods generally involve lysing the cells with a chaotropic (e.g., guanidinium isothiocyanate) and / or detergent (e.g., N-lauroyl sarcosine) prior to implementing processes for isolating particular populations of RNA. Methods may involve the use of organic solvents and / or alcohol to isolate nucleic acids. In some embodiments, the amount of alcohol added to a cell lysate achieves an alcohol concentration of about 55% to 60%. While different alcohols can be employed, ethanol works well. A solid support may be any structure, and it includes beads, filters, and columns, which may include a mineral or polymer support with electronegative groups. A glass fiber filter or column is effective for such isolation procedures. A nucleic acid isolation processes may sometimes include: a) lysing cells in the sample with a lysing solution comprising guanidinium, where a lysate with a concentration of at least about 1 M guanidinium is produced; b) extracting nucleic acid molecules from the lysate with an extraction solution comprising phenol; c) adding to the lysate an alcohol solution to form a lysate / alcohol mixture, wherein the concentration of alcohol in the mixture is between about 35% to about 70%; d) applying the lysate / alcohol mixture to a solid support; e) eluting the nucleic acid molecules from the solid support with an ionic solution; and, f) capturing the nucleic acid molecules. The sample may be dried down and resuspended in a liquid and volume appropriate for subsequent manipulation. Methods of Gene Transfer In order to mediate the effect of the transgene expression in a cell, it will be necessary to transfer the expression constructs into a cell. Such transfer may employ viral or non-viral methods of gene transfer. This section provides a discussion of methods and compositions of gene transfer. A transformed cell comprising an expression vector is generated by introducing into the cell the expression vector. Suitable methods for polynucleotide delivery for transformation of an organelle, a cell, a tissue or an organism for use with the current methods include virtually any method by which a polynucleotide (e.g., DNA) can be introduced into an organelle, a cell, a tissue or an organism. A host cell can, and has been, used as a recipient for vectors. Host cells may be derived from prokaryotes or eukaryotes, depending upon whether the desired result is replication of the vector or expression of part or all of the vector-encoded polynucleotide sequences. Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials. An appropriate host may be determined. Generally this is based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Bacterial cells used as host cells for vector replication and / or expression include DH5alpha, JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURE® Competent Cells and SOLOPACK Gold Cells (STRATAGENE®, La Jolla, CA). Alternatively, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to yeast, insects and mammals. Examples of mammalian eukaryotic host cells for replication and / or expression of a vector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC12. Examples of yeast strains include, but are not limited to, YPH499, YPH500 and YPH501. Nucleic acid vaccines may include, for example, non-viral DNA vectors, "naked" DNA and RNA, and viral vectors. Methods of transforming cells with these vaccines, and for optimizing the expression of genes included in these vaccines are known and are also discussed herein. Examples of Methods of Nucleic Acid or Viral Vector Transfer Any appropriate method may be used to transfect or transform the cells, or to administer the nucleotide sequences or compositions of the present methods. Certain examples are presented herein, and further include methods such as delivery using cationic polymers, lipid like molecules, and certain commercial products such as, for example, IN-VIVO-JET PEI. Ex vivo Transformation Various methods are available for transfecting vascular cells and tissues removed from an organism in an ex vivo setting. For example, canine endothelial cells have been genetically altered by retroviral gene transfer in vitro and transplanted into a canine (Wilson et al., Science, 244:1344- 1346, 1989). In another example, Yucatan minipig endothelial cells were transfected by retrovirus in vitro and transplanted into an artery using a double-balloon catheter (Nabel et al., Science, 244(4910):1342-1344, 1989). Thus, it is contemplated that cells or tissues may be removed and transfected ex vivo using the polynucleotides presented herein. In particular aspects, the transplanted cells or tissues may be placed into an organism. Injection In certain embodiments, an antigen presenting cell or a nucleic acid or viral vector may be delivered to an organelle, a cell, a tissue or an organism via one or more injections (i.e., a needle injection), such as, for example, subcutaneous, intradermal, intramuscular, intravenous, intraprotatic, intratumor, intraperitoneal, etc. Methods of injection include, for example, injection of a composition comprising a saline solution. Further embodiments include the introduction of a polynucleotide by direct microinjection. The amount of the expression vector used may vary upon the nature of the antigen as well as the organelle, cell, tissue or organism used. Intradermal, intranodal, or intralymphatic injections are some of the more commonly used methods of DC administration. Intradermal injection is characterized by a low rate of absorption into the bloodstream but rapid uptake into the lymphatic system. The presence of large numbers of Langerhans dendritic cells in the dermis will transport intact as well as processed antigen to draining lymph nodes. Proper site preparation is necessary to perform this correctly (i.e., hair is clipped in order to observe proper needle placement). Intranodal injection allows for direct delivery of antigen to lymphoid tissues. Intralymphatic injection allows direct administration of DCs. Electroporation In certain embodiments, a polynucleotide is introduced into an organelle, a cell, a tissue or an organism via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. In some variants of this method, certain cell wall- degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells (U.S. Patent No. 5,384,253). Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes (Potter et al., (1984) Proc. Nat'l Acad. Sci. USA, 81, 7161-7165), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., (1986) Mol. Cell Biol., 6,716-718) in this manner. In vivo electroporation for vaccines, or eVac, is clinically implemented through a simple injection technique. A DNA vector encoding a polypeptide is injected intradermally in a patient. Then electrodes apply electrical pulses to the intradermal space causing the cells localized there, especially resident dermal dendritic cells, to take up the DNA vector and express the encoded polypeptide. These polypeptide-expressing cells activated by local inflammation can then migrate to lymph-nodes, presenting antigens, for example. A nucleic acid is electroporetically administered when it is administered using electroporation, following, for example, but not limited to, injection of the nucleic acid or any other means of administration where the nucleic acid may be delivered to the cells by electroporation Methods of electroporation are discussed in, for example, Sardesai, N.Y., and Weiner, D.B., Current Opinion in Immunotherapy 23:421-9 (2011) and Ferraro, B. et al., Human Vaccines 7:120- 127 (2011). Calcium Phosphate In other embodiments, a polynucleotide is introduced to the cells using calcium phosphate precipitation. Human KB cells have been transfected with adenovirus 5 DNA (Graham and van der Eb, (1973) Virology, 52,456-467) using this technique. Also in this manner, mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al., Mol. Cell Biol., 10:689-695, 1990). DEAE-Dextran In another embodiment, a polynucleotide is delivered into a cell using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, T.V., Mol Cell Biol. 1985 May;5(5):1188-90). Sonication Loading Additional embodiments include the introduction of a polynucleotide by direct sonic loading. LTK- fibroblasts have been transfected with the thymidine kinase gene by sonication loading (Fechheimer et al., (1987) Proc. Nat'l Acad. Sci. USA, 84,8463-8467). Liposome-Mediated Transfection In a further embodiment, a polynucleotide may be entrapped in a lipid complex such as, for example, a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, (1991) In: Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands. pp. 87-104). Also contemplated is a polynucleotide complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen). Receptor Mediated Transfection Still further, a polynucleotide may be delivered to a target cell via receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in a target cell. In view of the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity. Certain receptor-mediated gene targeting vehicles comprise a cell receptor-specific ligand and a polynucleotide-binding agent. Others comprise a cell receptor-specific ligand to which the polynucleotide to be delivered has been operatively attached. Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, (1987) J. Biol. Chem., 262,4429-4432; Wagner et al., Proc. Natl. Acad. Sci. USA, 87(9):3410-3414, 1990; Perales et al., Proc. Natl. Acad. Sci. USA, 91:4086-4090, 1994; Myers, EPO 0273085), which establishes the operability of the technique. Specific delivery in the context of another mammalian cell type has been discussed (Wu and Wu, Adv. Drug Delivery Rev., 12:159-167, 1993). In certain aspects, a ligand is chosen to correspond to a receptor specifically expressed on the target cell population. In other embodiments, a polynucleotide delivery vehicle component of a cell-specific polynucleotide-targeting vehicle may comprise a specific binding ligand in combination with a liposome. The polynucleotide(s) to be delivered are housed within the liposome and the specific binding ligand is functionally incorporated into the liposome membrane. The liposome will thus specifically bind to the receptor(s) of a target cell and deliver the contents to a cell. Such systems have been shown to be functional using systems in which, for example, epidermal growth factor (EGF) is used in the receptor-mediated delivery of a polynucleotide to cells that exhibit upregulation of the EGF receptor. In still further embodiments, the polynucleotide delivery vehicle component of a targeted delivery vehicle may be a liposome itself, which may, for example, comprise one or more lipids or glycoproteins that direct cell-specific binding. For example, lactosyl-ceramide, a galactose-terminal asialoganglioside, have been incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes (Nicolau et al., (1987) Methods Enzymol., 149,157-176). It is contemplated that the tissue-specific transforming constructs may be specifically delivered into a target cell in a similar manner. Microprojectile Bombardment Microprojectile bombardment techniques can be used to introduce a polynucleotide into at least one, organelle, cell, tissue or organism (U.S. Patent No. 5,550,318; U.S. Patent No. 5,538,880; U.S. Patent No. 5,610,042; and PCT Application WO 94 / 09699). This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., (1987) Nature, 327, 70-73). There are a wide variety of microprojectile bombardment techniques known in the art, many of which are applicable to the present methods. In this microprojectile bombardment, one or more particles may be coated with at least one polynucleotide and delivered into cells by a propelling force. Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., (1990) Proc. Nat'l Acad. Sci. USA, 87, 9568-9572). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold particles or beads. Exemplary particles include those comprised of tungsten, platinum, and, in certain examples, gold, including, for example, nanoparticles. It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. However, it is contemplated that particles may contain DNA rather than be coated with DNA. DNA-coated particles may increase the level of DNA delivery via particle bombardment but are not, in and of themselves, necessary. Examples of Methods of Viral Vector-Mediated Transfer Any viral vector suitable for administering nucleotide sequences, or compositions comprising nucleotide sequences, to a cell or to a subject, such that the cell or cells in the subject may express the genes encoded by the nucleotide sequences may be employed in the present methods. In certain embodiments, a transgene is incorporated into a viral particle to mediate gene transfer to a cell. Typically, the virus simply will be exposed to the appropriate host cell under physiologic conditions, permitting uptake of the virus. The present methods are advantageously employed using a variety of viral vectors, as discussed below. Adenovirus Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized DNA genome, ease of manipulation, high titer, wide target-cell range, and high infectivity. The roughly 36 kb viral genome is bounded by 100-200 base pair (bp) inverted terminal repeats (ITR), in which are contained cis-acting elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome that contain different transcription units are divided by the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shut off (Renan, M. J. (1990) Radiother Oncol., 19, 197-218). The products of the late genes (L1, L2, L3, L4 and L5), including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP (located at 16.8 map units) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5' tripartite leader (TL) sequence, which makes them useful for translation. In order for adenovirus to be optimized for gene therapy, it is necessary to maximize the carrying capacity so that large segments of DNA can be included. It also is very desirable to reduce the toxicity and immunologic reaction associated with certain adenoviral products. The two goals are, to an extent, coterminous in that elimination of adenoviral genes serves both ends. By practice of the present methods, it is possible to achieve both these goals while retaining the ability to manipulate the therapeutic constructs with relative ease. The large displacement of DNA is possible because the cis elements required for viral DNA replication all are localized in the inverted terminal repeats (ITR) (100-200 bp) at either end of the linear viral genome. Plasmids containing ITR's can replicate in the presence of a non-defective adenovirus (Hay, R.T., et al., J Mol Biol. 1984 Jun 5;175(4):493-510). Therefore, inclusion of these elements in an adenoviral vector may permits replication. In addition, the packaging signal for viral encapsulation is localized between 194-385 bp (0.5-1.1 map units) at the left end of the viral genome (Hearing et al., J. (1987) Virol., 67,2555-2558). This signal mimics the protein recognition site in bacteriophage lambda DNA where a specific sequence close to the left end, but outside the cohesive end sequence, mediates the binding to proteins that are required for insertion of the DNA into the head structure. E1 substitution vectors of Ad have demonstrated that a 450 bp (0-1.25 map units) fragment at the left end of the viral genome could direct packaging in 293 cells (Levrero et al., Gene, 101:195-202, 1991). Previously, it has been shown that certain regions of the adenoviral genome can be incorporated into the genome of mammalian cells and the genes encoded thereby expressed. These cell lines are capable of supporting the replication of an adenoviral vector that is deficient in the adenoviral function encoded by the cell line. There also have been reports of complementation of replication deficient adenoviral vectors by "helping" vectors, e.g., wild-type virus or conditionally defective mutants. Replication-deficient adenoviral vectors can be complemented, in trans, by helper virus. This observation alone does not permit isolation of the replication-deficient vectors, however, since the presence of helper virus, needed to provide replicative functions, would contaminate any preparation. Thus, an additional element was needed that would add specificity to the replication and / or packaging of the replication-deficient vector. That element derives from the packaging function of adenovirus. It has been shown that a packaging signal for adenovirus exists in the left end of the conventional adenovirus map (Tibbetts et. al. (1977) Cell, 12,243-249). Later studies showed that a mutant with a deletion in the E1A (194-358 bp) region of the genome grew poorly even in a cell line that complemented the early (E1A) function (Hearing and Shenk, (1983) J. Mol. Biol. 167,809-822). When a compensating adenoviral DNA (0-353 bp) was recombined into the right end of the mutant, the virus was packaged normally. Further mutational analysis identified a short, repeated, position- dependent element in the left end of the Ad5 genome. One copy of the repeat was found to be sufficient for efficient packaging if present at either end of the genome, but not when moved toward the interior of the Ad5 DNA molecule (Hearing et al., J. (1987) Virol., 67, 2555-2558). By using mutated versions of the packaging signal, it is possible to create helper viruses that are packaged with varying efficiencies. Typically, the mutations are point mutations or deletions. When helper viruses with low efficiency packaging are grown in helper cells, the virus is packaged, albeit at reduced rates compared to wild-type virus, thereby permitting propagation of the helper. When these helper viruses are grown in cells along with virus that contains wild-type packaging signals, however, the wild-type packaging signals are recognized preferentially over the mutated versions. Given a limiting amount of packaging factor, the virus containing the wild-type signals is packaged selectively when compared to the helpers. If the preference is great enough, stocks approaching homogeneity may be achieved. To improve the tropism of ADV constructs for particular tissues or species, the receptor-binding fiber sequences can often be substituted between adenoviral isolates. For example the Coxsackie- adenovirus receptor (CAR) ligand found in adenovirus 5 can be substituted for the CD46-binding fiber sequence from adenovirus 35, making a virus with greatly improved binding affinity for human hematopoietic cells. The resulting "pseudotyped" virus, Ad5f35, has been the basis for several clinically developed viral isolates. Moreover, various biochemical methods exist to modify the fiber to allow re-targeting of the virus to target cells. Methods include use of bifunctional antibodies (with one end binding the CAR ligand and one end binding the target sequence), and metabolic biotinylation of the fiber to permit association with customized avidin-based chimeric ligands. Alternatively, one could attach ligands (e.g. anti-CD205 by heterobifunctional linkers (e.g. PEG- containing), to the adenovirus particle. Retrovirus The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, (1990) In: Virology, ed., New York: Raven Press, pp. 1437-1500). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes - gag, pol and env - that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene, termed psi, functions as a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and also are required for integration in the host cell genome (Coffin, 1990). Thus, for example, the present technology includes, for example, cells whereby the polynucleotide used to transduce the cell is integrated into the genome of the cell. In order to construct a retroviral vector, a nucleic acid encoding a promoter is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol and env genes but without the LTR and psi components is constructed (Mann et al., (1983) Cell, 33,153-159). When a recombinant plasmid containing a human cDNA, together with the retroviral LTR and psi sequences is introduced into this cell line (by calcium phosphate precipitation for example), the psi sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas, J.F., and Rubenstein, J.L.R., (1988) In: Vectors: a Survey of Molecular Cloning Vectors and Their Uses, Rodriquez and Denhardt, Eds.). Nicolas and Rubenstein; Temin et al., (1986) In: Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188; Mann et al., 1983). The media containing the recombinant retroviruses is collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression of many types of retroviruses require the division of host cells (Paskind et al., (1975) Virology, 67,242-248). An approach designed to allow specific targeting of retrovirus vectors recently was developed based on the chemical modification of a retrovirus by the chemical addition of galactose residues to the viral envelope. This modification could permit the specific infection of cells such as hepatocytes via asialoglycoprotein receptors, may be desired. A different approach to targeting of recombinant retroviruses was designed, which used biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor. The antibodies were coupled via the biotin components by using streptavidin (Roux et al., (1989) Proc. Nat'l Acad. Sci. USA, 86, 9079-9083). Using antibodies against major histocompatibility complex class I and class II antigens, the infection of a variety of human cells that bore those surface antigens was demonstrated with an ecotropic virus in vitro (Roux et al., 1989). Adeno-associated Virus AAV utilizes a linear, single-stranded DNA of about 4700 base pairs. Inverted terminal repeats flank the genome. Two genes are present within the genome, giving rise to a number of distinct gene products. The first, the cap gene, produces three different virion proteins (VP), designated VP-1, VP-2 and VP-3. The second, the rep gene, encodes four non-structural proteins (NS). One or more of these rep gene products is responsible for transactivating AAV transcription. The three promoters in AAV are designated by their location, in map units, in the genome. These are, from left to right, p5, p19 and p40. Transcription gives rise to six transcripts, two initiated at each of three promoters, with one of each pair being spliced. The splice site, derived from map units 42-46, is the same for each transcript. The four non-structural proteins apparently are derived from the longer of the transcripts, and three virion proteins all arise from the smallest transcript. AAV is not associated with any pathologic state in humans. Interestingly, for efficient replication, AAV requires "helping" functions from viruses such as herpes simplex virus I and II, cytomegalovirus, pseudorabies virus and, of course, adenovirus. The best characterized of the helpers is adenovirus, and many "early" functions for this virus have been shown to assist with AAV replication. Low-level expression of AAV rep proteins believed to hold AAV structural expression in check, and helper virus infection is thought to remove this block. The terminal repeats of the AAV vector can be obtained by restriction endonuclease digestion of AAV or a plasmid such as p201, which contains a modified AAV genome (Samulski et al., J. Virol., 61:3096-3101 (1987)), or by other methods, including but not limited to chemical or enzymatic synthesis of the terminal repeats based upon the published sequence of AAV. It can be determined, for example, by deletion analysis, the minimum sequence or part of the AAV ITRs which is required to allow function, i.e., stable and site-specific integration. It can also be determined which minor modifications of the sequence can be tolerated while maintaining the ability of the terminal repeats to direct stable, site-specific integration. AAV-based vectors have proven to be safe and effective vehicles for gene delivery in vitro, and these vectors are being developed and tested in pre-clinical and clinical stages for a wide range of applications in potential gene therapy, both ex vivo and in vivo (Carter and Flotte, (1995) Ann. N.Y. Acad. Sci., 770; 79-90; Chatteijee, et al., (1995) Ann. N.Y. Acad. Sci., 770,79-90; Ferrari et al., (1996) J. Virol., 70,3227-3234; Fisher et al., (1996) J. Virol., 70,520-532; Flotte et al., Proc. Nat'l Acad. Sci. USA, 90,10613-10617, (1993); Goodman et al. (1994), Blood, 84,1492-1500; Kaplitt et al., (1994) Nat'l Genet., 8,148-153; Kaplitt, M.G., et al., Ann Thorac Surg. 1996 Dec;62(6):1669-76; Kessler et al., (1996) Proc. Nat'l Acad. Sci. USA, 93,14082-14087; Koeberl et al., (1997) Proc. Nat'l Acad. Sci. USA, 94,1426-1431; Mizukami et al., (1996) Virology, 217,124-130). AAV-mediated efficient gene transfer and expression in the lung has led to clinical trials for the treatment of cystic fibrosis (Carter and Flotte, 1995; Flotte et al., Proc. Nat'l Acad. Sci. USA, 90, 10613-10617, (1993)). Similarly, the prospects for treatment of muscular dystrophy by AAV- mediated gene delivery of the dystrophin gene to skeletal muscle, of Parkinson's disease by tyrosine hydroxylase gene delivery to the brain, of hemophilia B by Factor IX gene delivery to the liver, and potentially of myocardial infarction by vascular endothelial growth factor gene to the heart, appear promising since AAV-mediated transgene expression in these organs has recently been shown to be highly efficient (Fisher et al., (1996) J. Virol., 70,520-532; Flotte et al., 1993; Kaplitt et al., 1994; 1996; Koeberl et al., 1997; McCown et al., (1996) Brain Res., 713,99-107; Ping et al., (1996) Microcirculation, 3,225-228; Xiao et al., (1996) J. Virol., 70,8098-8108). Other Viral Vectors Other viral vectors are employed as expression constructs in the present methods and compositions. Vectors derived from viruses such as vaccinia virus (Ridgeway, (1988) In: Vectors: A survey of molecular cloning vectors and their uses, pp. 467-492; Baichwal and Sugden, (1986) In, Gene Transfer, pp. 117-148; Coupar et al., Gene, 68:1-10, 1988) canary poxvirus, and herpes viruses are employed. These viruses offer several features for use in gene transfer into various mammalian cells. Once the construct has been delivered into the cell, the nucleic acid encoding the transgene are positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the transgene is stably integrated into the genome of the cell. This integration is in the cognate location and orientation via homologous recombination (gene replacement) or it is integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid is stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed. Methods for Treating a Disease The present methods also encompass methods of treatment or prevention of a disease where administration of cells by, for example, infusion, may be beneficial. Cells, such as, for example, progenitor cells, such as, for example, hematopoietic stem cells, mesenchymal stromal cells, stem cells, pluripotent stem cells, and embryonic stem cells may be used for cell therapy. The cells may be from a donor, or may be cells obtained from the patient. The cells may, for example, be used in regeneration, for example, to replace the function of diseased cells. The cells may also be modified to express a heterologous gene so that biological agents may be delivered to specific microenvironments such as, for example, diseased bone marrow or metastatic deposits. Mesenchymal stromal cells have also, for example, been used to provide immunosuppressive activity, and may be used in the treatment of graft versus host disease and autoimmune disorders. The cells provided in the present application contain a safety switch that may be valuable in a situation where following cell therapy, the cells need to be removed. For example, where progenitor cells are provided to the patient, in some situations there may be an adverse event, such as inappropriate differentiation of the cell into a more mature cell type, or an undesired invitation into another tissue, for example, where it is necessary to remove the therapeutic cells. By "therapeutic cell" is meant a cell used for cell therapy, that is, a cell administered to a subject to treat or prevent a condition or disease. In such cases, where the cells have a negative effect, the present methods may be used to remove the therapeutic cells through partial apoptosis. In other examples, T cells are used to treat various diseases and conditions, and as a part of stem cell transplantation. An adverse event that may occur after haploidentical T cell transplantation is graft versus host disease (GvHD). The likelihood of GvHD occurring increases with the increased number of T cells that are transplanted. This limits the number of T cells that may be infused. By having the ability to selectively remove the infused T cells in the event of GvHD in the patient, a greater number of T cells may be infused, increasing the number to greater than 10<com:Sup>6< / com:Sup>, greater than 10<com:Sup>7< / com:Sup>, greater than 10<com:Sup>8< / com:Sup>, or greater than 10<com:Sup>9< / com:Sup> cells. The number of T cells / kg body weight that may be administered may be, for example, from about 1 x 10<com:Sup>4< / com:Sup> T cells / kg body weight to about 9 x 10<com:Sup>7< / com:Sup> T cells / kg body weight, for example about 1, 2, 3, 4, 5, 6, 7, 8, or 9 x 10<com:Sup>4< / com:Sup>; about 1, 2, 3, 4, 5, 6, 7, 8, or <com:Math><semantics>9×105<annotation encoding="application / x-tex">9 \times 10^5< / annotation>< / semantics>< / com:Math>; about 1, 2, 3, 4, 5, 6, 7, 8, or <com:Math><semantics>9×106<annotation encoding="application / x-tex">9 \times 10^6< / annotation>< / semantics>< / com:Math>; or about 1, 2, 3, 4, 5, 6, 7, 8, or <com:Math><semantics>9×107<annotation encoding="application / x-tex">9 \times 10^7< / annotation>< / semantics>< / com:Math> T cells / kg body weight. In other examples, therapeutic cells other than T cells may be used. The number of therapeutic cells / kg body weight that may be administered may be, for example, from about 1 x 104 T cells / kg body weight to about 9 x 10<com:Sup>7< / com:Sup> T cells / kg body weight, for example about 1, 2, 3, 4, 5, 6, 7, 8, or <com:Math><semantics>9×104<annotation encoding="application / x-tex">9 \times 10^4< / annotation>< / semantics>< / com:Math>; about 1, 2, 3, 4, 5, 6, 7, 8, or <com:Math><semantics>9×105<annotation encoding="application / x-tex">9 \times 10^5< / annotation>< / semantics>< / com:Math>; about 1, 2, 3, 4, 5, 6, 7, 8, or <com:Math><semantics>9×106<annotation encoding="application / x-tex">9 \times 10^6< / annotation>< / semantics>< / com:Math>; or about 1, 2, 3, 4, 5, 6, 7, 8, or <com:Math><semantics>9×107<annotation encoding="application / x-tex">9 \times 10^7< / annotation>< / semantics>< / com:Math> therapeutic cells / kg body weight. The term "unit dose" as it pertains to the inoculum refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of pharmaceutical composition calculated to produce the desired immunogenic effect in association with the required diluent. The specifications for the unit dose of an inoculum are dictated by and are dependent upon the unique characteristics of the pharmaceutical composition and the particular immunologic effect to be achieved. An effective amount of the pharmaceutical composition, such as the multimeric ligand presented herein, would be the amount that achieves this selected result of selectively removing the desired number or concentration of cells that include the caspase-9 vector, such that over 60%, 70%, 80%, 85%, 90%, 95%, or 97%, or that under 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the caspase-9 expressing cells are killed. The term is also synonymous with "sufficient amount." The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular composition being administered, the size of the subject, and / or the severity of the disease or condition. One can empirically determine the effective amount of a particular composition presented herein without necessitating undue experimentation. The terms "contacted" and "exposed," when applied to a cell, tissue or organism, are used herein to describe the process by which the pharmaceutical composition and / or another agent, such as for example a chemotherapeutic or radiotherapeutic agent, are delivered to a target cell, tissue or organism or are placed in direct juxtaposition with the target cell, tissue or organism. To achieve cell killing or stasis, the pharmaceutical composition and / or additional agent(s) are delivered to one or more cells in a combined amount effective to kill the cell(s) or prevent them from dividing. The administration of the pharmaceutical composition may precede, be co-current with and / or follow the other agent(s) by intervals ranging from minutes to weeks. In embodiments where the pharmaceutical composition and other agent(s) are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the times of each delivery, such that the pharmaceutical composition and agent(s) would still be able to exert an advantageously combined effect on the cell, tissue or organism. For example, in such instances, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i.e., within less than about a minute) with the pharmaceutical composition. In other aspects, one or more agents may be administered within of from substantially simultaneously, about 1 minute, to about 24 hours to about 7 days to about 1 to about 8 weeks or more, and any range derivable therein, prior to and / or after administering the expression vector. Yet further, various combination regimens of the pharmaceutical composition presented herein and one or more agents may be employed. Optimized and Personalized Therapeutic Treatment The induction of apoptosis after administration of the dimer may be optimized by determining the stage of graft versus host disease, or the number of undesired therapeutic cells that remain in the patient. For example, determining that a patient has GvHD, and the stage of the GvHD, provides an indication to a clinician that it may be necessary to induce caspase-9 associated apoptosis by administering the multimeric ligand. In another example, determining that a patient has a reduced level of GvHD after treatment with the multimeric ligand may indicate to the clinician that no additional dose of the multimeric ligand is needed. Similarly, after treatment with the multimeric ligand, determining that the patient continues to exhibit GvHD symptoms, or suffers a relapse of GvHD may indicate to the clinician that it may be necessary to administer at least one additional dose of multimeric ligand. The term "dosage" is meant to include both the amount of the dose and the frequency of administration, such as, for example, the timing of the next dose. The term "dosage level" refers to the amount of the multimeric ligand administered in relation to the body weight of the subject. Thus increasing the dosage level would mean increasing the amount of the ligand administered relative to the subject's weight. In addition, increasing the concentration of the dose administered, such as, for example, when the multimeric ligand is administered using a continuous infusion pump would mean that the concentration administered (and thus the amount administered) per minute, or second, is increased. An indication of adjusting or maintaining a subsequent drug dose, such as, for example, a subsequence dose of the multimeric ligand, and / or the subsequent drug dosage, can be provided in any convenient manner. An indication may be provided in tabular form (e.g., in a physical or electronic medium) in some embodiments. For example, the graft versus host disease observed symptoms may be provided in a table, and a clinician may compare the symptoms with a list or table of stages of the disease. The clinician then can identify from the table an indication for subsequent drug dose. In certain embodiments, an indication can be presented (e.g., displayed) by a computer, after the symptoms or the GvHD stage is provided to the computer (e.g., entered into memory on the computer). For example, this information can be provided to a computer (e.g., entered into computer memory by a user or transmitted to a computer via a remote device in a computer network), and software in the computer can generate an indication for adjusting or maintaining a subsequent drug dose, and / or provide the subsequent drug dose amount. Once a subsequent dose is determined based on the indication, a clinician may administer the subsequent dose or provide instructions to adjust the dose to another person or entity. The term "clinician" as used herein refers to a decision maker, and a clinician is a medical professional in certain embodiments. A decision maker can be a computer or a displayed computer program output in some embodiments, and a health service provider may act on the indication or subsequent drug dose displayed by the computer. A decision maker may administer the subsequent dose directly (e.g., infuse the subsequent dose into the subject) or remotely (e.g., pump parameters may be changed remotely by a decision maker). Methods as presented herein include without limitation the delivery of an effective amount of an activated cell, a nucleic acid, or an expression construct encoding the same. An "effective amount" of the pharmaceutical composition, generally, is defined as that amount sufficient to detectably and repeatedly to achieve the stated desired result, for example, to ameliorate, reduce, minimize or limit the extent of the disease or its symptoms. Other more rigorous definitions may apply, including elimination, eradication or cure of disease. In some embodiments there may be a step of monitoring the biomarkers to evaluate the effectiveness of treatment and to control toxicity. Formulations and Routes for Administration to Patients Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions—expression constructs, expression vectors, fused proteins, transfected or transduced cells, in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals. The multimeric ligand, such as, for example, AP1903, may be delivered, for example at doses of about 0.1 to 10 mg / kg subject weight, of about 0.1 to 5 mg / kg subject weight, of about 0.2 to 4 mg / kg subject weight, of about 0.3 to 3 mg / kg subject weight, of about 0.3 to 2 mg / kg subject weight, or about 0.3 to 1 mg / kg subject weight, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 mg / kg subject weight. In some embodiments, the ligand is provided at 0.4mg / kg per dose, for example at a concentration of 5mg / mL. Vials or other containers may be provided containing the ligand at, for example, a volume per vial of about 0.25 ml to about 10 ml, for example, about 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 ml, for example, about 2 ml. One may generally desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells. Buffers also may be employed when recombinant cells are introduced into a patient. Aqueous compositions comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. A pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is known. Except insofar as any conventional media or agent is incompatible with the vectors or cells, its use in the rapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. The active compositions may include classic pharmaceutical preparations. Administration of these compositions will be via any common route so long as the target tissue is available via that route. This includes, for example, oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, discussed herein. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form is sterile and is fluid to the extent that easy syringability exists. It is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In certain examples, isotonic agents, for example, sugars or sodium chloride may be included. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. For oral administration, the compositions may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient also may be dispersed in dentifrices, including, for example: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include, for example, water, binders, abrasives, flavoring agents, foaming agents, and humectants. The compositions may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include, for example, the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution may be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media can be employed. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570- 1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations may meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologics standards. Examples The examples set forth below illustrate certain embodiments and do not limit the technology. Mechanisms for selectively ablating the donor cells have been studied as safety switches for cellular therapies, but there have been complications. Some experience with safety-switch genes to date has been in T lymphocytes since immunotherapy with these cells has proved efficacious as treatment for viral infections and malignancies (Walter, E.A., et al., N. Engl. J. Med. 1995, 333:1038-44; Rooney, C.M., et al., Blood. 1998, 92:1549-55; Dudley, M.E., et al., Science 2002, 298:850-54; Marjit, W.A., et al., Proc. Natl. Acad. Sci. USA 2003, 100:2742-47). The herpes simplex virus I-derived thymidine kinase (HSVTK) gene has been used as an in vivo suicide switch in donor T-cell infusions to treat recurrent malignancy and Epstein Barr virus (EBV) lymphoproliferation after hematopoietic stem cell transplantation (Bonini C, et al., Science. 1997, 276:1719-1724; Tiberghien P, et al., Blood. 2001, 97:63-72). However, destruction of T cells causing graft-versus-host disease was incomplete, and the use of gancyclovir (or analogs) as a pro-drug to activate HSV-TK precludes administration of gancyclovir as an antiviral drug for cytomegalovirus infections. This mechanism of action also requires interference with DNA synthesis, relying on cell division, so that cell killing may be protracted over several days and incomplete, producing a lengthy delay in clinical benefit (Ciceri, F., et al., Lancet Oncol. 2009, 262:1019-24). Moreover, HSV-TK-directed immune responses have resulted in elimination of HSV-TK-transduced cells, even in immunosuppressed human immunodeficiency virus and bone marrow transplant patients, compromising the persistence and hence efficacy of the infused T cells. HSV-TK is also virus-derived, and therefore potentially immunogenic (Bonini C, et al., Science. 1997, 276:1719-1724; Riddell SR, et al., Nat Med. 1996, 2:216-23). The E coli-derived cytosine deaminase gene has also been used clinically (Freytag SO, et al., Cancer Res. 2002, 62:4968-4976), but as a xenoantigen it may be immunogenic and thus incompatible with T-cell- based therapies that require long-term persistence. Transgenic human CD20, which can be activated by a monoclonal chimeric anti-CD20 antibody, has been proposed as a nonimmunogenic safety system (Introna M, et al., Hum Gene Ther. 2000, 11: 611-620). The following section provides examples of method of providing a safety switch in cells used for cellular therapy, using a caspase-9 chimeric protein. Example 1: Construction and Evaluation of caspase-9 Suicide Switch Expression Vectors Vector construction and confirmation of expression A safety switch that can be stably and efficiently expressed in human T cells is presented herein. The system includes human gene products with low potential immunogenicity that have been modified to interact with a small molecule dimerizer drug that is capable of causing the selective elimination of transduced T cells expressing the modified gene. Additionally the inducible caspase- 9 maintains function in T cells overexpressing antiapoptotic molecules. Expression vectors suitable for use as a therapeutic agent were constructed that included a modified human caspase-9 activity fused to a human FK506 binding protein (FKBP), such as, for example, FKBP12v36. The caspase-9 / FK506 hybrid activity can be dimerized using a small molecule pharmaceutical. Full length, truncated, and modified versions of the caspase-9 activity were fused to the ligand binding domain, or multimerization region, and inserted into the retroviral vector MSCV.IRES.GRP, which also allows expression of the fluorescent marker, GFP. FIG. 1A illustrates the full length, truncated and modified caspase-9 expression vectors constructed and evaluated as a suicide switch for induction of apoptosis. The full-length inducible caspase-9 molecule (F'-F-C-Casp9) includes 2, 3, or more FK506 binding proteins (FKBPs—for example, FKBP12v36 variants) linked with a Gly-Ser-Gly-Gly-Gly-Ser linker to the small and large subunit of the Caspase molecule (see FIG. 1A). Full-length inducible caspase-9 (F'F-C-Casp9.I.GFP) has a full-length caspase-9, also includes a Caspase recruitment domain (CARD; GenBank NM001 229) linked to 2 12-kDa human FK506 binding proteins (FKBP12; GenBank AH002 818) that contain an F36V mutation (FIG. 1A). The amino acid sequence of one or more of the FKBPs (F') was codon-wobbled (e.g., the 3<com:Sup>rd< / com:Sup> nucleotide of each amino acid codon was altered by a silent mutation that maintained the originally encoded amino acid) to prevent homologous recombination when expressed in a retrovirus. F'F-C-Casp9C3S includes a cysteine to serine mutation at position 287 that disrupts its activation site. In constructs F'F-Casp9, F-C-Casp9, and F'-Casp9, either the Caspase activation domain (CARD), one FKBP, or both, were deleted, respectively. All constructs were cloned into MSCV.IRES.GFP as EcoRI- Xhol fragments. 293T cells were transfected with each of these constructs and 48 hours after transduction expression of the marker gene GFP was analyzed by flow cytometry. In addition, 24 hours after transfection, 293T cells were incubated overnight with 100 nM CID and subsequently stained with the apoptosis marker annexin V. The mean and standard deviation of transgene expression level (mean GFP) and number of apoptotic cells before and after exposure to the chemical inducer of dimerization (CID) (% annexin V within GFP~ cells) from 4 separate experiments are shown in the second through fifth columns of the table in FIG. 1A. In addition to the level of GFP expression and staining for annexin V, the expressed gene products of the full length, truncated and modified caspase-9 were also analyzed by western blot to confirm the caspase-9 genes were being expressed and the expressed product was the expected size. The results of the western blot are presented in FIG. 1B. Coexpression of the inducible caspase-9 constructs of the expected size with the marker gene GFP in transfected 293T cells was demonstrated by Western blot using a caspase-9 antibody specific for amino acid residues 299-318, present both in the full-length and truncated Caspase molecules as well as a GFP-specific antibody. Western blots were performed as presented herein. Transfected 293T cells were resuspended in lysis buffer (50% Tris / Gly, 10% sodium dodecyl sulfate [SDS], 4% beta-mercaptoethanol, 10% glycerol, 12% water, 4% bromophenol blue at 0.5%) containing aprotinin, leupeptin, and phenylmethylsulfonyl fluoride (Boehringer, Ingelheim, Germany) and incubated for 30 minutes on ice. After a 30-minute centrifugation, supernatant was harvested, mixed 1:2 with Laemmli buffer (Bio-Rad, Hercules, CA), boiled and loaded on a 10% SDS-polyacrylamide gel. The membrane was probed with rabbit anti-caspase-9 (amino acid residues 299-3 18) immunoglobulin G (IgG; Affinity BioReagents, Golden, CO; 1:500 dilution) and with mouse anti-GFP lgG (Covance, Berkeley, CA; 1:25,000 dilution). Blots were then exposed to appropriate peroxidase-coupled secondary antibodies and protein expression was detected with enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL). The membrane was then stripped and reprobed with goat polyclonal antiactin (Santa Cruz Biotechnology; 1:500 dilution) to check equality of loading. Additional smaller size bands, seem in FIG. 1B, likely represent degradation products. Degradation products for the F'F-C-Casp9 and F'F-Casp9 constructs may not be detected due to a lower expression level of these constructs as a result of their basal activity. Equal loading of each sample was confirmed by the substantially equal amounts of actin shown at the bottom of each lane of the western blot, indicating substantially similar amounts of protein were loaded in each lane. Evaluation of caspase-9 suicide switch expression constructs. Cell lines B 95-8 EBV transformed B-cell lines (LCLs), Jurkat, and MT-2 cells (kindly provided by Dr S. Marriott, Baylor College of Medicine, Houston, TX) were cultured in RPMI 1640 (Hyclone, Logan, UT) containing 10% fetal bovine serum (FBS; Hyclone). Polyclonal EBV-specific T-cell lines were cultured in 45% RPMI / 45% Clicks (Irvine Scientific, Santa Ana, CA) / 10% FBS and generated as previously reported. Briefly, peripheral blood mononuclear cells (2 x 10<com:Sup>6< / com:Sup> per well of a 24-well plate) were stimulated with autologous LCLs irradiated at 4000 rads at a responder-to-stimulator (R / S) ratio of 40:1. After 9 to 12 days, viable cells were restimulated with irradiated LCLs at an R / S ratio of 4:1. Subsequently, cytotoxic T cells (CTLs) were expanded by weekly restimulation with LCLs in the presence of 40 U / mL to 100 U / mL recombinant human interleukin-2 (rhIL-2; Proleukin; Chiron, Emeryville, CA). Retrovirus transduction For the transient production of retrovirus, 293T cells were transfected with iCasp9 / iFas constructs, along with plasmids encoding gag-pol and RD 114 envelope using GeneJuice transfection reagent (Novagen, Madison, WI). Virus was harvested 48 to 72 hours after transfection, snap frozen, and stored at ~80 ℃ until use. A stable FLYRD 18-derived retroviral producer line was generated by multiple transductions with VSV-G pseudotyped transient retroviral supernatant. FLYRD18 cells with highest transgene expression were single-cell sorted, and the clone that produced the highest virus titer was expanded and used to produce virus for lymphocyte transduction. The transgene expression, function, and retroviral titer of this clone were maintained during continuous culture for more than 8 weeks. For transduction of human lymphocytes, a non-tissue-culture-treated 24-well plate (Becton Dickinson, San Jose, CA) was coated with recombinant fibronectin fragment (FN CH- 296; Retronectin; Takara Shuzo, Otsu, Japan; 4 μg / mL in PBS, overnight at 4°C) and incubated twice with 0.5 mL retrovirus per well for 30 minutes at 37°C. Subsequently, 3 x10° to 5 x 10° T cells per well were transduced for 48 to 72 hours using 1 mL virus per well in the presence of 100 U / mL IL-2. Transduction efficiency was determined by analysis of expression of the coexpressed marker gene green fluorescent protein (GFP) on a FACScan flow cytometer (Becton Dickinson). For functional studies, transduced CTLs were either non-selected or segregated into populations with low, intermediate, or high GFP expression using a MoFlo cytometer (Dako Cytomation, Ft Collins, CO) as indicated. Induction and analysis of apoptosis CID (AP20187; ARIAD Pharmaceuticals) at indicated concentrations was added to transfected 293T cells or transduced CTLs. Adherent and nonadherent cells were harvested and washed with annexin binding buffer (BD Pharmingen, San Jose, CA). Cells were stained with annexin-V and 7- amino-actinomycin D (7-AAD) for 15 minutes according to the manufacturer's instructions (BD Pharmingen). Within 1 hour after staining, cells were analyzed by flow cytometry using CellQuest software (Becton Dickinson). Cytotoxicity assay The cytotoxic activity of each CTL line was evaluated in a standard 4-hour <com:Sup>51< / com:Sup>Cr release assay, as previously presented. Target cells included autologous LCLs, human leukocyte antigen (HLA) class I-mismatched LCLs and the lymphokine-activated killer cell-sensitive T-cell lymphoma line HSB-2. Target cells incubated in complete medium or 1% Triton X-100 (Sigma, St Louis, MO) were used to determine spontaneous and maximum <com:Sup>51< / com:Sup>Cr release, respectively. The mean percentage of specific lysis of triplicate wells was calculated as 100 X (experimental release - spontaneous release) / (maximal release - spontaneous release). Phenotyping Cell-surface phenotype was investigated using the following monoclonal antibodies: CD3, CD4, CD8 (Becton Dickinson) and CD56 and TCR-<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> / <com:Math><semantics>β<annotation encoding="application / x-tex">\beta< / annotation>< / semantics>< / com:Math> (Immunotech, Miami, FL). <com:Math><semantics>△<annotation encoding="application / x-tex">\triangle< / annotation>< / semantics>< / com:Math>NGFR-iFas was detected using anti-NGFR antibody (Chromaprobe, Aptos, CA). Appropriate matched isotype controls (Becton Dickinson) were used in each experiment. Cells were analyzed with a FACSscan flow cytometer (Becton Dickinson). Analysis of cytokine production The concentration of interferon-<com:Math><semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>< / com:Math> (IFN-<com:Math><semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>< / com:Math>), IL-2, IL-4, IL-5, IL-10, and tumor necrosis factor- <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> (TNFα) in CTL culture supernatants was measured using the Human Th1 / Th2 cytokine cytometric Bead Array (BD Pharmingen) and the concentration of IL-12 in the culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN) according to the instructions of the manufacturer. In vivo experiments Non-obese diabetic severe combined immunodeficient (NOD / SCID) mice, 6 to 8 weeks of age, were irradiated (250 rad) and injected subcutaneously in the right flank with <com:Math><semantics>10×106<annotation encoding="application / x-tex">10 \times 10^6< / annotation>< / semantics>< / com:Math> to <com:Math><semantics>15×106<annotation encoding="application / x-tex">15 \times 10^6< / annotation>< / semantics>< / com:Math> LCLs resuspended in Matrigel (BD Bioscience). Two weeks later mice bearing tumors that were approxi- mately 0.5 cm in diameter were injected into the tail vein with a 1:1 mixture of nontransduced and iCasp9.I.GFPhigh-transduced EBV CTLs (total 15 x 10<com:Sup>6< / com:Sup>). At 4 to 6 hours prior and 3 days after CTL infusion, mice were injected intraperitoneally with recombinant hlL-2 (2000 U; Proleukin; Chiron). On day 4, the mice were randomly segregated in 2 groups: 1 group received CID (50 µg AP20187, intraperitoneally) and 1 group received carrier only (16.7% propanedial, 22.5% PEG400, and 1.25% Tween 80, intraperitoneally). On day 7, all mice were killed. Tumors were homoge- nized and stained with antihuman CD3 (BD Pharmingen). By FACS analysis, the number of GFP+ cells within the gated CD3<com:Sup>+< / com:Sup> population was evaluated. Tumors from a control group of mice that received only nontransduced CTLs (total <com:Math><semantics>15×106<annotation encoding="application / x-tex">15 \times 10^6< / annotation>< / semantics>< / com:Math>) were used as a negative control in the analysis of CD3<com:Sup>+< / com:Sup> / GFP<com:Sup>+< / com:Sup> cells. Optimization of expression and function of inducible caspase-9 Caspases 3, 7, and 9 were screened for their suitability as inducible safety-switch molecules both in transfected 293T cells and in transduced human T cells. Only inducible caspase-9 (iCasp9) was expressed at levels sufficient to confer sensitivity to the chosen CID (e.g., chemical inducer of dimerization). An initial screen indicated that the full length iCasp9 could not be maintained stably at high levels in T cells, possibly due to transduced cells being eliminated by the basal activity of the transgene. The CARD domain is involved in physiologic dimerization of caspase-9 molecules, by a cytochrome C and adenosine triphosphate (ATP)—driven interaction with apoptotic protease- activating factor 1 (Apaf-1). Because of the use of a CID to induce dimerization and activation of the suicide switch, the function of the CARD domain is superfluous in this context and removal of the CARD domain was investigated as a method of reducing basal activity. Given that only dimerization rather than multimerization is required for activation of caspase-9, a single FKBP12v36 domain also was investigated as a method to effect activation. The activity of the resultant truncated and / or modified forms of caspase-9 (e.g., the CARD domain, or one of the 2 FKBP domains, or both, are removed) were compared. A construct with a disrupted activation site, F'F-C-Casp9<com:Sub>c->s< / com:Sub>, provided a nonfunctional control (see FIG. 1A). All constructs were cloned into the retroviral vector MSCV<com:Sup>26< / com:Sup> in which retroviral long terminal repeats (LTRs) direct transgene expression and enhanced GFP is coexpressed from the same mRNA by use of an internal ribosomal entry site (IRES). In transfected 293T cells, expression of all inducible caspase-9 constructs at the expected size as well as coexpression of GFP was demonstrated by Western blot (see FIG. 1B). Protein expression (estimated by mean fluorescence of GFP and visualized on Western blot) was highest in the nonfunctional construct F'F-C-Casp9<com:Sub>C->S< / com:Sub> and greatly diminished in the full-length construct F'F-C-Casp9. Removal of the CARD (F'F-Casp9), one FKBP (F-C-Casp9), or both (F-Casp9) resulted in progressively higher expression of both inducible caspase-9 and GFP, and correspondingly enhanced sensitivity to CID (see FIG. 1A). Based on these results, the F-Casp9 construct (henceforth referred to as iCasp9<com:Sub>M< / com:Sub>) was used for further study in human T lymphocytes. Stable expression of iCasp9<com:Sub>M< / com:Sub> in human T lymphocytes The long-term stability of suicide gene expression is of utmost importance, since suicide genes must be expressed for as long as the genetically engineered cells persist. For T-cell transduction, a FLYRD18-derived retroviral producer clone that produces high-titer RD114-pseudotyped virus was generated to facilitate the transduction of T cells. iCasp9<com:Sub>M< / com:Sub> expression in EBV-specific CTL lines (EBV-CTL) was evaluated since EBV-specific CTL lines have well-characterized function and specificity and are already being used as in vivo therapy for prevention and treatment of EBV- associated malignancies. Consistent transduction efficiencies of EBV-CTLs of more than 70% (mean, 75.3%; range, 71.4%-83.0% in 5 different donors) were obtained after a single transduction with retrovirus. The expression of iCasp9<com:Sub>M< / com:Sub> in EBV-CTLs was stable for at least 4 weeks after transduction without selection or loss of transgene function. iCasp9<com:Sub>M< / com:Sub> does not alter transduced T-cell characteristics To ensure that expression of iCasp9<com:Sub>M< / com:Sub> did not alter T-cell characteristics, the phenotype, antigen- specificity, proliferative potential, and function of nontransduced or nonfunctional iCasp9<com:Sub>C->S< / com:Sub>- transduced EBV-CTLs was compared with that of iCasp9<com:Sub>M< / com:Sub>-transduced EBV-CTLs. In 4 separate donors, transduced and nontransduced CTLs consisted of equal numbers of CD4<com:Sup>+< / com:Sup>, CD8<com:Sup>+< / com:Sup>, CD56<com:Sup>+< / com:Sup>, and TCR <com:Math><semantics>α / β<annotation encoding="application / x-tex">\alpha / \beta< / annotation>< / semantics>< / com:Math> <com:Sup>+< / com:Sup> cells (see FIG. 2A). Similarly, production of cytokines including IFN-<com:Math><semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>< / com:Math>, TNF<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>, IL-10, IL-4, IL-5, and IL-2 was unaltered by iCasp9<com:Sub>M< / com:Sub> expression (see FIG. 2B). iCasp9<com:Sub>M< / com:Sub>-transduced EBV-- CTLs specifically lysed autologous LCLs comparable to nontransduced and control-transduced CTLs (see FIG. 2C). Expression of iCasp9M did not affect the growth characteristics of exponentially growing CTLs, and importantly, dependence on antigen and IL-2 for proliferation was preserved (see FIG. 2D). FIGS. 2A and 2B graphically phenotypic and secretion data of type TH1 and TH2 cytokines upon antigen stimulation. FIG. 2C graphically illustrates the level of cytotoxic activity against autologous EBV-transformed lymphoblastoid B-cell line (LCL), HLA-mismatched LCL, and HSB-2 (a LAK cell target) were compared in nontransduced (white bars), F-Casp9<com:Sub>M< / com:Sub>- transduced (black bars), and FF-C-Casp9<com:Sub>C->S< / com:Sub>-transduced (stippled bars) EBV-specific CTLs (EBV-CTLs) on day 15 to day 18 after transduction (2 antigenic stimulations after transduction). The mean and standard deviation of triplicate wells are shown. Examples of experiments using EBV-CTLs from 4 different donors are shown. FIG. 2D graphically illustrates the antigen dependence of iCasp9<com:Sub>M< / com:Sub>-transduced CTLs. On day 21 after transduction the normal weekly antigenic stimulation with autologous LCLs and IL-2 was continued (black diamonds) or discontinued (black squares). Discontinuation of antigen stimulation resulted in a steady decline of T cells. Elimination of more than 99% of T lymphocytes selected for high transgene expression in vitro Inducible iCasp9<com:Sub>M< / com:Sub> proficiency in CTLs was tested by monitoring loss of GFP-expressing cells after administration of CID; 91.3% (range, 89.5%-92.6% in 5 different donors) of GFP+ cells were eliminated after a single 10-nM dose of CID (see FIG. 3A). Similar results were obtained regardless of exposure time to CID (range, 1 hour-continuous). In all experiments, CTLs that survived CID treatment had low transgene expression with a 70% (range, 55%-82%) reduction in mean fluorescence intensity of GFP after CID. No further elimination of the surviving GFP<com:Sup>+< / com:Sup>T cells could be obtained by an antigenic stimulation followed by a second 10-nM dose of CID. Therefore, the non-responding CTLs most likely expressed insufficient iCasp9<com:Sub>M< / com:Sub> for functional activation by CID. To investigate the correlation between low levels of expression and CTL non-response to CID, CTLs were sorted for low, intermediate, and high expression of the linked marker gene GFP and mixed 1:1 with nontransduced CTLs from the same donor to allow for an accurate quantitation of the number of transduced T cells responding to CID-induced apoptosis. The number of transduced T cells eliminated increased with the level of GFP transgene expression (see FIGS. 4A, 4B and 4C). To determine the correlation between transgene expression and function of iCasp9<com:Sub>M< / com:Sub>, iCasp9<com:Sub>M< / com:Sub> IRES.GFP-transduced EBV-CTL were selected for low (mean 21), intermediate (mean 80) and high (mean 189) GFP expression (see FIG. 4A). Selected T-cells were incubated overnight with 10 nM CID and subsequently stained with annexin V and 7-AAD. Indicated are the percentages of annexin <com:Math><semantics>V++ / 7<annotation encoding="application / x-tex">V^++ / 7< / annotation>< / semantics>< / com:Math>-AAD- and annexin <com:Math><semantics>V+ / 7<annotation encoding="application / x-tex">V^+ / 7< / annotation>< / semantics>< / com:Math>-AAD+ T-cells (see FIG. 4B). Selected T-cells were mixed 1:1 with non-transduced T-cells and incubated with 10 nM CID following antigenic stimulation. Indicated is the percentage of residual GFP-positive T-cells on day 7 (see FIG. 4C). For GFP<com:Sub>high< / com:Sub>-selected cells, 10 nM CID led to deletion of 99.1% (range, 98.7%-99.4%) of transduced cells (see FIG. 3A). On the day of antigen stimulation, F-Casp9<com:Sub>M< / com:Sub>.I.GFP-transduced CTLs were either untreated or treated with 10 nM CID. Seven days later, the response to CID was measured by flow cytometry for GFP. The percentage of transduced T cells was adjusted to 50% to allow for an accurate measurement of residual GFP+ cells after CID treatment. The responses to CID in unselected (top row of FIG. 3A) and GFP<com:Sub>high< / com:Sub>-selected CTLs (bottom row of FIG. 3A) was compared. The percentage of residual GFP+ cells is indicated (see FIG. 3A). Rapid induction of apoptosis in the GFP<com:Sub>high< / com:Sub>-selected cells is demonstrated by apoptotic characteristics such as cell shrinkage and fragmentation within 14 hours of CID administration (see FIG. 3B). After overnight incubation with 10 nM CID, F-Casp9<com:Sub>M< / com:Sub>.I.GFP<com:Sub>high< / com:Sub>-transduced T cells had apoptotic characteristics such as cell shrinkage and fragmentation by microscopic evaluation. Of the T cells selected for high expression, 64% (range, 59%-69%) had an apoptotic (annexin-V<com:Sup>+< / com:Sup>+ / 7- AAD and 30% (range, 26%-32%) had a necrotic (annexinV / 7-AAD) phenotype (see FIG. 3C). Staining with markers of apoptosis showed that 64% of T cells had an apoptotic phenotype (annexin <com:Math><semantics>V+<annotation encoding="application / x-tex">V^+< / annotation>< / semantics>< / com:Math>, 7-AAD<com:Math><semantics>‾<annotation encoding="application / x-tex">\bar{}< / annotation>< / semantics>< / com:Math>, lower right quadrant) and 32% a necrotic phenotype (annexin <com:Math><semantics>V+<annotation encoding="application / x-tex">V^+< / annotation>< / semantics>< / com:Math>, 7-AAD<com:Math><semantics>‾<annotation encoding="application / x-tex">\bar{}< / annotation>< / semantics>< / com:Math>, upper right quadrant). A representative example of 3 separate experiments is shown. In contrast, the induction of apoptosis was significantly lower in T cells selected for intermediate or low GFP expression (see FIGS. 4A, 4B and 4C). For clinical applications therefore, versions of the expression constructs with selectable markers that allow selection for high copy number, high levels of expression, or both high copy number and high levels of expression may be desirable. CID-induced apoptosis was inhibited by the panCaspase inhibitor zVAD-fmk (100 µM for 1 hour prior to adding CID. Titration of CID showed that 1 nM CID was sufficient to obtain the maximal deletion effect (FIG. 3D). A dose-response curve using the indicated amounts of CID (AP20187) shows the sensitivity of F-Casp9<com:Sub>M< / com:Sub>.I.GFP<com:Sub>high< / com:Sub> to CID. Survival of GFP<com:Sup>+< / com:Sup> cells is measured on day 7 after administration of the indicated amount of CID. The mean and standard deviation for each point are given. Similar results were obtained using another chemical inducer of dimerization (CID), AP1903, which was clinically shown to have substantially no adverse effects when administered to healthy volunteers. The dose response remained unchanged for at least 4 weeks after transduction. iCasp9<com:Sub>M< / com:Sub> is functional in malignant cells that express antiapoptotic molecules caspase-9 was selected as an inducible proapoptotic molecule for clinical use rather than previously presented iFas and iFADD, because caspase-9 acts relatively late in apoptosis signaling and therefore is expected to be less susceptible to inhibition by apoptosis inhibitors. Thus, suicide function should be preserved not only in malignant, transformed T-cell lines that express antiapoptotic molecules, but also in subpopulations of normal T cells that express elevated antiapoptotic molecules as part of the process to ensure long-term preservation of memory cells. To further investigate the hypothesis, the function of iCasp9<com:Sub>M< / com:Sub> and iFas was first compared in EBV-CTLs. To eliminate any potential vector based difference, inducible Fas also was expressed in the MSCV.IRES.GFP vector, like iCasp9. For these experiments both △NGFR.iFas.I.GFP and iCasp9<com:Sub>M< / com:Sub>.I.GFP-transduced CTLs were sorted for GFP<com:Sub>high< / com:Sub> expression and mixed with nontransduced CTLs at a 1:1 ratio to obtain cell populations that expressed either iFas or iCasp9<com:Sub>M< / com:Sub> at equal proportions and at similar levels (see FIG. 5A). EBV-CTLs transduced with △NGFR-iFas.I.GFP are shown in the left panel of FIG. 5A. EBV-CTLs transduced with iCasp9<com:Sub>M< / com:Sub>.I.GFP are shown in the right panel of FIG. 5A. The EBV-CTLs were sorted for high GFP expression and mixed 1:1 with nontransduced CTLs as presented. The percentages of <com:Math><semantics>△<annotation encoding="application / x-tex">\triangle< / annotation>< / semantics>< / com:Math>NGFR<com:Sup>+< / com:Sup> / GFP<com:Sup>+< / com:Sup> and GFP<com:Sup>+< / com:Sup>T cells are indicated. Elimination of GFP<com:Sup>+< / com:Sup> cells after administration of 10 nM CID was more rapid and more efficient in iCasp9<com:Sub>M< / com:Sub> than in iFas-transduced CTLs (99.2% + / - 0.14% of iCasp9<com:Sub>M< / com:Sub>-transduced cells compared with 89.3% + / - 4.9% of iFas-transduced cells at day 7 after CID; P < .05; see FIG. 5B). On the day of LCL stimulation, 10 nM CID was administered, and GFP was measured at the time points indicated to determine the response to CID. Black diamonds represent data for <com:Math><semantics>△<annotation encoding="application / x-tex">\triangle< / annotation>< / semantics>< / com:Math>NGFR- iFas.I.GFP; black squares represent data for iCasp9<com:Sub>M< / com:Sub>.I.GFP. Mean and standard deviation of 3 experiments are shown. The function of iCasp9M and iFas was also compared in 2 malignant T-cell lines: Jurkat, an apoptosis-sensitive T-cell leukemia line, and MT-2, an apoptosis-resistant T-cell line, due to c-FLIP and bcl-xL expression. Jurkat cells and MT-2 cells were transduced with iFas and iCasp9<com:Sub>M< / com:Sub> with similar efficiencies (92% vs 84% in Jurkat, 76% vs 70% in MT-2) and were cultured in the presence of 10 nM CID for 8 hours. annexin-V staining showed that although iFas and iCasp9<com:Sub>M< / com:Sub> induced apoptosis in an equivalent number of Jurkat cells (56.4% + / - 15.6% and 57.2% + / -18.9%, respectively), only activation of iCasp9<com:Sub>M< / com:Sub> resulted in apoptosis of MT-2 cells (19.3% + / - 8.4% and <com:Math><semantics>57.9%+−11.9%<annotation encoding="application / x-tex">57.9\% + - 11.9\%< / annotation>< / semantics>< / com:Math> for iFas and iCasp9<com:Sub>M< / com:Sub>, respectively; see Figure 5C). The human T-cell lines Jurkat (left) and MT-2 (right) were transduced with <com:Math><semantics>△<annotation encoding="application / x-tex">\triangle< / annotation>< / semantics>< / com:Math>NGFR-iFas.I.GFP (top row of FIG. 5C) or iCasp9<com:Sub>M< / com:Sub>.I.GFP (bottom row of FIG. 5C). An equal percentage of T cells were transduced with each of the suicide genes: 92% for △NGFR-iFas.l.GFP versus 84% for iCasp9<com:Sub>M< / com:Sub>.I.GFP in Jurkat, and 76% for <com:Math><semantics>△<annotation encoding="application / x-tex">\triangle< / annotation>< / semantics>< / com:Math>NGFR-iFas.I.GFP versus 70% for iCasp9<com:Sub>M< / com:Sub>.I.GFP in MT-2. T cells were either nontreated or incubated with 10 nM CID. Eight hours after exposure to CID, apoptosis was measured by staining for annexin V and 7-AAD. A representative example of 3 experiments is shown. PE indicates phycoerythrin. These results demonstrate that in T cells overexpressing apoptosis-inhibiting molecules, the function of iFas can be blocked, while iCasp9<com:Sub>M< / com:Sub> can still effectively induce apoptosis. iCasp9M-mediated elimination of T cells expressing an immunomodulatory transgene To determine whether iCasp9M could effectively destroy cells genetically modified to express an active transgene product, the ability of iCasp9<com:Sub>M< / com:Sub> to eliminate EBV-CTLs stably expressing IL-12 was measured. While IL- 12 was undetectable in the supernatant of nontransduced and iCasp9<com:Sub>M< / com:Sub>.IRES.GFP-transduced CTLs, the supernatant of iCasp9<com:Sub>M< / com:Sub>.IRES.IL-12-transduced cells contained 324 pg / mL to 762 pg / mL IL-12. After administration of 10 nM CID, however, the IL-12 in the supernatant fell to undetectable levels (< 8 pg / mL). Thus, even without prior sorting for high transgene expressing cells, activation of iCasp9<com:Sub>M< / com:Sub> is sufficient to completely eliminate all T cells producing biologically relevant levels of IL-12 (Figure 6). The function of iCasp9M when coexpressed with IL-12 is graphically represented by bar graphs in FIG. 6. The marker gene GFP in the iCasp9<com:Sub>M< / com:Sub>.I.GFP constructs was replaced by flexi IL-12, encoding the p40 and p35 subunits of human IL-12. iCasp9<com:Sub>M-< / com:Sub>I.GFP- and iCasp9<com:Sub>M-< / com:Sub>I.IL-12—transduced EBV-CTLs were stimulated with LCLs, and then left untreated or exposed to 10 nM CID. Three days after a second antigenic stimulation, the levels of IL-12 in the culture supernatant were measured by IL-12 ELISA (detection limit of this assay is 8 pg / mL). The mean and standard deviation of triplicate wells are indicated. Results of 1 of 2 experiments with CTLs from 2 different donors are shown. Elimination of more than 99% of T cells selected for high transgene expression in vivo The function of iCasp9<com:Sub>M< / com:Sub> also was evaluated in transduced EBV-CTLs in vivo. A SCID mouse— human xenograft model was used for adoptive immunotherapy. After intravenous infusion of a 1:1 mixture of nontransduced and iCasp9<com:Sub>M< / com:Sub>.IRES.GFP<com:Sub>high< / com:Sub>-transduced CTLs into SCID mice bearing an autologous LCL xenograft, mice were treated either with a single dose of CID or carrier only. Three days after CID / carrier administration, tumors were analyzed for human CD3<com:Sup>+< / com:Sup> / GFP<com:Sup>+< / com:Sup> cells. Detection of the nontransduced component of the infusion product, using human anti-CD3 antibodies, confirmed the success of the tail-vein infusion in mice that received CID. In mice treated with CID, there was more than a 99% reduction in the number of human CD3<com:Sup>+< / com:Sup> / GFP<com:Sup>+< / com:Sup> T cells, compared with infused mice treated with carrier alone, demonstrating equally high sensitivity of iCasp9<com:Sub>M< / com:Sub>-transduced T cells in vivo and in vitro (see FIG. 7). The function of iCasp9<com:Sub>M< / com:Sub> in vivo, is graphically illustrated in FIG. 7. NOD / SCID mice were irradiated and injected subcutaneously with <com:Math><semantics>10×106<annotation encoding="application / x-tex">10 \times 10^6< / annotation>< / semantics>< / com:Math> to <com:Math><semantics>15×106<annotation encoding="application / x-tex">15 \times 10^6< / annotation>< / semantics>< / com:Math> LCLs. After 14 days, mice bearing tumors of 0.5cm in diameter received a total of 15 x10<com:Sup>6< / com:Sup> EBV-CTLs (50% of these cells were nontransduced and 50% were transduced with iCasp9<com:Sub>M< / com:Sub>.I.GFP and sorted for high GFP expression). On day 3 after CTL administration, mice received either CID (50 μg AP20187; (black diamonds, n=6) or carrier only (black squares, n=5) and on day 6 the presence of human CD3+ / GFP+T cells in the tumors was analyzed. Human CD3<com:Sup>+< / com:Sup> T cells isolated from the tumors of a control group of mice that received only nontransduced CTLs (15 <com:Math><semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics>< / com:Math>10<com:Sup>6< / com:Sup> CTLs; n= 4) were used as a negative control for the analysis of CD3<com:Sup>+< / com:Sup> / GFP<com:Sup>+< / com:Sup> T cells within the tumors. Discussion Presented herein are expression vectors expressing suicide genes suitable for eliminating gene- modified T cells in vivo, in some embodiments. Suicide gene expression vectors presented herein have certain non-limiting advantageous features including stable coexpression in all cells carrying the modifying gene, expression at levels high enough to elicit cell death, low basal activity, high specific activity, and minimal susceptibility to endogenous antiapoptotic molecules. Presented herein, in certain embodiments, is an inducible caspase-9, iCasp9<com:Sub>M< / com:Sub>, which has low basal activity allowing stable expression for more than 4 weeks in human T cells. A single 10-nM dose of a small molecule chemical inducer of dimerization (CID) is sufficient to kill more than 99% of iCasp9<com:Sub>M< / com:Sub>-transduced cells selected for high transgene expression both in vitro and in vivo. Moreover, when coexpressed with Th1 cytokine IL-12, activation of iCasp9<com:Sub>M< / com:Sub> eliminated all detectable IL-12-producing cells, even without selection for high transgene expression. caspase-9 acts downstream of most antiapoptotic molecules, therefore a high sensitivity to CID is preserved regardless of the presence of increased levels of antiapoptotic molecules of the bcl-2 family. Thus, iCasp9<com:Sub>M< / com:Sub> also may prove useful for inducing destruction even of transformed T cells and memory T cells that are relatively resistant to apoptosis. Unlike other Caspase molecules, proteolysis does not appear sufficient for activation of caspase-9. Crystallographic and functional data indicate that dimerization of inactive caspase-9 monomers leads to conformational change—induced activation. The concentration of pro-caspase-9, in a physiologic setting, is in the range of about 20 nM, well below the threshold needed for dimerization. Without being limited by theory, it is believed the energetic barrier to dimerization can be overcome by homophilic interactions between the CARD domains of Apaf-1 and caspase-9, driven by cytochrome C and ATP. Overexpression of caspase-9 joined to 2 FKBPs may allow spontaneous dimerization to occur and can account for the observed toxicity of the initial full length caspase-9 construct. A decrease in toxicity and an increase in gene expression was observed following removal of one FKBP, most likely due to a reduction in toxicity associated with spontaneous dimerization. While multimerization often is involved in activation of surface death receptors, dimerization of caspase-9 should be sufficient to mediate activation. Data presented herein indicates that iCasp9 constructs with a single FKBP function as effectively as those with 2 FKBPs. Increased sensitivity to CID by removal of the CARD domain may represent a reduction in the energetic threshold of dimerization upon CID binding. The persistence and function of virus- or bacteria-derived lethal genes, such as HSV-TK and cytosine deaminase, can be impaired by unwanted immune responses against cells expressing the virus or bacteria derived lethal genes. The FKBPs and proapoptotic molecules that form the components of iCasp9<com:Sub>M< / com:Sub> are human-derived molecules and are therefore less likely to induce an immune response. Although the linker between FKBP and caspase-9 and the single point mutation in the FKBP domain introduce novel amino acid sequences, the sequences were not immunologically recognized by macaque recipients of iFas-transduced T cells. Additionally, because the components of iCasp9<com:Sub>M< / com:Sub> are human-derived molecules, no memory T cells specific for the junction sequences should be present in a recipient, unlike virus-derived proteins such as HSV-TK, thereby reducing the risk of immune response—mediated elimination of iCasp9<com:Sub>M< / com:Sub>-transduced T cells. Previous studies using inducible Fas or the death effector domains (DED) of Fas associated death domain proteins (FADD) showed that approximately 10% of transduced cells were unresponsive to activation of the destructive gene. As observed in experiments presented here, a possible explanation for unresponsiveness to CID is low expression of the transgene. The iCasp9<com:Sub>M< / com:Sub>- transduced T cells in our study and iFas-transduced T cells in studies by others that survived after CID administration had low levels of transgene expression. In an attempt to overcome a perceived retroviral "positional effect", increased levels of homogeneous expression of the transgene were achieved by flanking retroviral integrants with the chicken beta-globin chromatin insulator. Addition of the chromatin insulator dramatically increased the homogeneity of expression in transduced 293T cells, but had no significant effect in transduced primary T cell. Selection of T cells with high expression levels minimized variability of response to the dimerizer. Over 99% of transduced T cells sorted for high GFP expression were eliminated after a single 10-nM CID dose. This demonstration supports the hypothesis that cells expressing high levels of suicide gene can be isolated using a selectable marker. A very small number of resistant residual cells may cause a resurgence of toxicity, a deletion efficiency of up to 2 logs will significantly decrease this possibility. For clinical use, coexpression with a nonimmunogenic selectable marker such as truncated human NGFR, CD20, or CD34 (e.g., instead of GFP) will allow for selection of high transgene—expressing T cells. Coexpression of the suicide switch (e.g., iCASP9<com:Sub>M< / com:Sub>) and a suitable selectable marker (e.g., truncated human NGFR, CD20, CD34, the like and combinations thereof) can be obtained using either an internal ribosome entry site (IRES) or posttranslational modification of a fusion protein containing a self-cleaving sequence (eg, 2A). In contrast, in situations where the sole safety concern is the transgene- mediated toxicity (eg, artificial T-cell receptors, cytokines, the like or combinations thereof), this selection step may be unnecessary, as tight linkage between iCasp9<com:Sub>M< / com:Sub> and transgene expression enables elimination of substantially all cells expressing biologically relevant levels of the therapeutic transgene. This was demonstrated by coexpressing iCasp9<com:Sub>M< / com:Sub> with IL-12. Activation of iCasp9<com:Sub>M< / com:Sub> substantially eliminated any measurable IL- 12 production. The success of transgene expression and subsequent activation of the "suicide switch" may depend on the function and the activity of the transgene. Another possible explanation for unresponsiveness to CID is that high levels of apoptosis inhibitors may attenuate CID-mediated apoptosis. Examples of apoptosis inhibitors include c-FLIP, bcl-2 family members and inhibitors of apoptosis proteins (IAPs), which normally regulate the balance between apoptosis and survival. For instance, upregulation of c-FLIP and bcl-2 render a subpopulation of T cells, destined to establish the memory pool, resistant to activation-induced cell death in response to cognate target or antigen-presenting cells. In several T-lymphoid tumors, the physiologic balance between apoptosis and survival is disrupted in favor of cell survival. A suicide gene should delete substantially all transduced T cells including memory and malignantly transformed cells. Therefore, the chosen inducible suicide gene should retain a significant portion if not substantially all of its activity in the presence of increased levels of antiapoptotic molecules. The apical location of iFas (or iFADD) in the apoptosis signaling pathway may leave it especially vulnerable to inhibitors of apoptosis, thus making these molecules less well suited to being the key component of an apoptotic safety switch. Caspase 3 or 7 would seem well suited as terminal effector molecules, however neither could be expressed at functional levels in primary human T cells. Therefore caspase-9, was chosen as the suicide gene, because capsase 9 functions late enough in the apoptosis pathway that it bypasses the inhibitory effects of c-FLIP and antiapoptotic bcl-2 family members, and caspase-9 also could be expressed stably at functional levels. Although X-linked inhibitor of apoptosis (XIAP) could in theory reduce spontaneous caspase-9 activation, the high affinity of AP20187 (or AP1903) for FKBP<com:Sub>V36< / com:Sub> may displace this noncovalently associated XIAP. In contrast to iFas, iCasp9<com:Sub>M< / com:Sub> remained functional in a transformed T-cell line that overexpresses antiapoptotic molecules, including bcl-xL. Presented herein is an inducible safety switch, designed specifically for expression from an oncoretroviral vector by human T cells. iCasp9<com:Sub>M< / com:Sub> can be activated by AP1903 (or analogs), a small chemical inducer of dimerization that has proven safe at the required dose for optimum deletional effect, and unlike ganciclovir or rituximab has no other biologic effects in vivo. Therefore, expression of this suicide gene in T cells for adoptive transfer can increase safety and also may broaden the scope of clinical applications. Example 2: Using the iCasp9 Suicide Gene to Improve the Safety of Allodepleted T Cells after Haploidentical Stem Cell Transplantation Presented in this example are expression constructs and methods of using the expression constructs to improve the safety of allodepleted T cells after haploidentical stem cell transplantation. A retroviral vector encoding iCasp9 and a selectable marker (truncated CD19) was generated as a safety switch for donor T cells. Even after allodepletion (using anti-CD25 immunotoxin), donor T cells could be efficiently transduced, expanded, and subsequently enriched by CD19 immunomagnetic selection to >90% purity. The engineered cells retained anti-viral specificity and functionality, and contained a subset with regulatory phenotype and function. Activating iCasp9 with a small-molecule dimerizer rapidly produced >90% apoptosis. Although transgene expression was downregulated in quiescent T cells, iCasp9 remained an efficient suicide gene, as expression was rapidly upregulated in activated (alloreactive) T cells. Materials and Methods Generation of allodepleted T cells Allodepleted cells were generated from healthy volunteers as previously presented. Briefly, peripheral blood mononuclear cells (PBMCs) from healthy donors were co-cultured with irradiated recipient Epstein Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) at responder-to-stimulator ratio of 40:1 in serum-free medium (AIM V; Invitrogen, Carlsbad, CA). After 72 hours, activated T cells that expressed CD25 were depleted from the co-culture by overnight incubation in RFT5-SMPT-dgA immunotoxin. Allodepletion was considered adequate if the residual CD3<com:Sup>+< / com:Sup>CD25<com:Sup>+< / com:Sup> population was <1% and residual proliferation by 3H- thymidine incorporation was <10%. Plasmid and retrovirus SFG.iCasp9.2A.CD19 consists of inducible caspase-9 (iCasp9) linked, via a cleavable 2A-like sequence, to truncated human CD19 (CD19; see FIG. 8A). iCasp9 consists of a human FK5 06-binding protein (FKBP12; GenBank AH002 818) with an F36V mutation, connected via a Ser- Gly-Gly-Gly-Ser linker to human caspase-9 (CASP9; GenBank NM 001229). The F36V mutation increases the binding affinity of FKBP12 to the synthetic homodimerizer, AP20187 or AP1903. The Caspase recruitment domain (CARD) has been deleted from the human caspase-9 sequence because its physiological function has been replaced by FKBP12, and its removal increases transgene expression and function. The 2A-like sequence encodes an 20 amino acid peptide from Thosea asigna insect virus, which mediates >99% cleavage between a glycine and terminal proline residue, resulting in 19 extra amino acids in the C terminus of iCasp9, and one extra proline residue in the N terminus of CD19. CD19 consists of full-length CD19 (GenBank NM 001770) truncated at amino acid 333 (TDPTRRF), which shortens the intracytoplasmic domain from 242 to 19 amino acids, and removes all conserved tyrosine residues that are potential sites for phosphorylation. A stable PG13 clone producing Gibbon ape leukemia virus (Gal-V) pseudotyped retrovirus was made by transiently transfecting Phoenix Eco cell line (ATCC product #SD3444; ATCC, Manassas, VA) with SFG.iCasp9.2A.CD19. This produced Eco-pseudotyped retrovirus. The PG13 packaging cell line (ATCC) was transduced three times with Eco-pseudotyped retrovirus to generate a producer line that contained multiple SFG.iCasp9.2A.CD19 proviral integrants per cell. Single cell cloning was performed, and the PG13 clone that produced the highest titer was expanded and used for vector production. Retroviral transduction Culture medium for T cell activation and expansion consisted of 45% RPMI 1640 (Hyclone, Logan, UT), 45% Clicks (Irvine Scientific, Santa Ana, CA) and 10% fetal bovine serum (FBS; Hyclone). Allodepleted cells were activated by immobilized anti-CD3 (OKT3; Ortho Biotech, Bridgewater, NJ) for 48 hours before transduction with retroviral vector (see FIG. 8B). FIG. 8B presents an overview of the process for production of the "final cell product" that expresses the transduced transgene. Selective allodepletion was performed by co-culturing donor PBMC with recipient EBV-LCL to activate alloreactive cells: activated cells expressed CD25 and were subsequently eliminated by anti-CD25 immunotoxin. The allodepleted cells were activated by OKT3 and transduced with the retroviral vector 48 hours later. Immunomagnetic selection was performed on day 4 of transduction; the positive fraction was expanded for a further 4 days and cryopreserved. In small-scale experiments, non-tissue culture-treated 24-well plates (Becton Dickinson, San Jose, CA) were coated with OKT3 1 g / ml for 2 to 4 hours at 37°C. Allodepleted cells were added at 1×10<com:Sup>6< / com:Sup> cells per well. At 24 hours, 100U / ml of recombinant human interleukin-2 (IL-2) (Proleukin; Chiron, Emeryville, CA) was added. Retroviral transduction was performed 48 hours after activation. Non-tissue culture-treated 24-well plates were coated with 3.5µg / cm<com:Sup>2< / com:Sup> recombinant fibronectin fragment (CH-296; Retronectin; Takara Mirus Bio, Madison, WI) and the wells loaded twice with retroviral vector-containing supernatant at 0.5ml per well for 30 minutes at 37 °C, following which OKT3 -activated cells were plated at 5 ×10<com:Sup>5< / com:Sup> cells per well in fresh retroviral vector-containing supernatant and T cell culture medium at a ratio of 3:1, supplemented with 100U / ml IL-2. Cells were harvested after 2 to 3 days and expanded in the presence of 50U / ml IL-2. Scaling-up production of gene-modified allodepleted cells Scale-up of the transduction process for clinical application used non-tissue culture-treated T75 flasks (Nunc, Rochester, NY), which were coated with 10ml of OKT3 1µg / ml or 10ml of fibronectin 7μg / ml at 4 °C overnight. Fluorinated ethylene propylene bags corona-treated for increased cell adherence (2PF-0072AC, American Fluoroseal Corporation, Gaithersburg, MD) were also used. Allodepleted cells were seeded in OKT3 -coated flasks at 1×10<com:Sup>6< / com:Sup> cells / ml. 100U / ml IL-2 was added the next day. For retroviral transduction, retronectin-coated flasks or bags were loaded once with 10ml of retrovirus-containing supernatant for 2 to 3 hours. OKT3- activated T cells were seeded at <com:Math><semantics>1×106<annotation encoding="application / x-tex">1 \times 10^6< / annotation>< / semantics>< / com:Math> cells / ml in fresh retroviral vector-containing medium and T cell culture medium at a ratio of 3:1, supplemented with 100U / ml IL-2. Cells were harvested the following morning and expanded in tissue-culture treated T75 or T175 flasks in culture medium supplemented with between about 50 to 100U / ml IL-2 at a seeding density of between about 5×10<com:Sup>5< / com:Sup> cells / ml to 8×10<com:Sup>5< / com:Sup> cells / ml. CD19 immunomagnetic selection Immunomagnetic selection for CD19 was performed 4 days after transduction. Cells were labeled with paramagnetic microbeads conjugated to monoclonal mouse anti-human CD19 antibodies (Miltenyi Biotech, Auburn, CA) and selected on MS or LS columns in small scale experiments and on a CliniMacs Plus automated selection device in large scale experiments. CD19-selected cells were expanded for a further 4 days and cryopreserved on day 8 post transduction. These cells were referred to as "gene-modified allodepleted cells". Immunophenotyping and pentamer analysis Flow cytometric analysis (FACSCalibur and CellQuest software; Becton Dickinson) was performed using the following antibodies: CD3, CD4, CD8, CD19, CD25, CD27, CD28, CD45RA, CD45RO, CD56 and CD62L. CD19-PE (Clone 4G7; Becton Dickinson) was found to give optimum staining and was used in all subsequent analysis. A Non-transduced control was used to set the negative gate for CD19. An HLA-pentamer, HLA-B8-RAKFKQLL (Proimmune, Springfield, VA) was used to detect T cells recognizing an epitope from EBV lytic antigen (BZLF1). HLA-A2-NLVPMVATV pentamer was used to detect T cells recognizing an epitope from CMV-pp65 antigen. Interferon- ELISpot assay for anti-viral response Interferon- ELISpot for assessment of responses to EBV, CMV and adenovirus antigens was performed using known methods. Gene-modified allodepleted cells cryopreserved at 8 days post transduction were thawed and rested overnigh...

Claims

1. Use of a multimeric ligand for controlling survival of transplanted therapeutic cells in a subject; wherein, the transplanted therapeutic cells comprise a nucleic acid that encodes a chimeric polypeptide comprising (a) a multimerization region and (b) a caspase-9 polypeptide or a modified caspase-9 polypeptide, wherein (i) the caspase-9 polypeptide or the modified caspase-9 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9; (ii) the modified caspase-9 polypeptide differs from SEQ ID NO: 9 by at least one amino acid substitution selected from the group consisting of S144A, S144D, Y153A, Y153F, S183A, S195A, S196A, S196D, S307A, D315A, A316G, T317A, T317C, T317E, T317S, P318A, F319A, F319W, F326K, D327G, D327K, D327R, Q328K, Q328R, L329E, L329G, L329K, D330A, D330E, D330G, D330N, D330S, D330V, A331K, C403A, C403S, C403T, F404T, F404W, F404Y, N405A, N405F, N405Q, N405T, F406A, F406T, F406W, F406Y, G402A, G402I, G402Q, G402Y, C403P, F404A, F404S, and F406L; (iii) the modified caspase-9 polypeptide has a reduced IC 50 and an elongated dose response curve in response to the multimeric ligand compared to a caspase-9 polypeptide that is not modified; and wherein the multimeric ligand binds to the multimerization region in an amount effective to kill less than 50% of transplanted therapeutic cells that express the caspase-9 polypeptide or the modified caspase-9 polypeptide.

2. The use of claim 1, wherein the multimeric ligand binds to the multimerization region in an amount effective to kill less than 40% of transplanted therapeutic cells that express the caspase-9 polypeptide or the modified caspase-9 polypeptide.

3. The use of claim 1 or 2, wherein the multimeric ligand binds to the multimerization region in an amount effective to kill less than 30% of transplanted therapeutic cells that express the caspase- 9 polypeptide or the modified caspase-9 polypeptide.

4. The use of any one of claims 1 to 3, wherein the therapeutic cells further express a heterologous protein.

5. The use of claim 4, wherein the heterologous protein is a chimeric antigen receptor.

6. Use of a multimeric ligand for controlling survival of transplanted therapeutic cells in a subject; wherein the transplanted therapeutic cells comprise, (a) a first subset of therapeutic cells that comprise a nucleic acid that encodes a chimeric polypeptide comprising a multimerization region and a first caspase-9 polypeptide, wherein the first caspase-9 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9; (b) a second subset of the therapeutic cells with a nucleic acid that encodes a chimeric polypeptide comprising a multimerization region and a second caspase-9 polypeptide, wherein (i) the second caspase-9 polypeptide is a modified caspase-9 polypeptide and comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9, and the modified caspase-9 polypeptide differs from SEQ ID NO: 9 by at least one amino acid substitution selected from the group consisting of S144A, S144D, Y153A, Y153F, S183A, S195A, S196A, S196D, S307A, D315A, A316G, T317A, T317C, T317E, T317S, P318A, F319A, F319W, F326K, D327G, D327K, D327R, Q328K, Q328R, L329E, L329G, L329K, D330A, D330E, D330G, D330N, D330S, D330V, A331K, C403A, C403S, C403T, F404T, F404W, F404Y, N405A, N405F, N405Q, N405T, F406A, F406T, F406W, F406Y, G402A, G402I, G402Q, G402Y, C403P, F404A, F404S, and F406L; (ii) the first caspase-9 polypeptide and the second modified caspase-9 polypeptide comprise different amino acid sequences and have different basal activities; (iii) the second modified caspase-9 polypeptide has a reduced IC 50 and an elongated dose response curve in response to the multimeric ligand compared to the first caspase-9 polypeptide that is not modified; and wherein the multimeric ligand binds to the multimerization region in an amount effective to kill more of the first subset of therapeutic cells than the second subset of therapeutic cells.

7. The use of claim 6, wherein the first or the second subset of therapeutic cells are selected from the group consisting of hematopoietic stem cells, inducible progenitor cells (iPS), embryonic stem (ES) cells, mesenchymal stem cells (MSCs), plasma (B) cells, myocytes, natural killer (NK) cells, macrophages, tumor infiltrating lymphocytes (TILs), and T cells.

8. The use of claim 6 or 7, wherein the first or the second subset of therapeutic cells are T cells.

9. The use of any one of claim 6 to 8, wherein the first or the second subset of therapeutic cells further express a chimeric antigen receptor.

10. The use of claim 8, wherein the T cells are for administration to the subject, and the T cells are not allodepleted before the administration.

11. The use of any one of claims 1 to 10, wherein the subject has cancer.

12. The use of any one of claims 1 to 10, wherein the subject has a solid tumor.

13. The use of claim 11, wherein the cancer is present in the blood or bone marrow of the subject.

14. The use of any one of claims 1 to 10, wherein the subject has a blood or bone marrow disease.

15. The use of any one of claims 1 to 10, wherein the subject has been diagnosed with any condition or disorder that can be alleviated by stem cell transplantation.

16. The use of any one of claims 1 to 10, wherein the subject has been diagnosed with sickle cell anemia or metachromatic leukodystrophy.

17. The use of any one of claims 1 to 10, wherein the subject has been diagnosed with a condition selected from the group consisting of a primary immune deficiency disorder, hemophagocytosis lymphohistiocytosis (HLH), an inherited marrow failure disorder, a hemoglobinopathy, a metabolic disorder, and an osteoclast disorder.

18. The use of any one of claims 1 to 10, wherein the subject has been diagnosed with a condition selected from the group consisting of Severe Combined Immune Deficiency (SCID), Combined Immune Deficiency (CID), Congenital T-cell Defect / Deficiency, Common Variable Immune Deficiency (CVID), Chronic Granulomatous Disease, IPEX (Immune deficiency, polyendocrinopathy, enteropathy, X-linked), Wiskott-Aldrich Syndrome, CD40 Ligand Deficiency, Leukocyte Adhesion Deficiency, DOCK 8 Deficiency, IL-10 Deficiency / IL-10 Receptor Deficiency, GATA 2 deficiency, X-linked lymphoproliferative disease (XLP), Cartilage Hair Hypoplasia, Shwachman Diamond Syndrome, Diamond Blackfan Anemia, Dyskeratosis Congenita, Fanconi Anemia, Congenital Neutropenia, Sickle Cell Disease, Thalassemia, Mucopolysaccharidosis, Sphingolipidoses, and Osteopetrosis.

19. The use of any one of claims 1 to 10, wherein the subject exhibits one or more Stage 1 graft versus host disease symptoms.

20. The use of any one of claims 1 to 10, wherein the subject exhibits one or more Stage 2 graft versus host disease symptoms.

21. The use of any one of claims 1 to 10, wherein the subject exhibits one or more Stage 3 graft versus host disease symptoms.

22. The use of any one of claims 1 to 10, wherein the subject exhibits one or more Stage 4 graft versus host disease symptoms.

23. The use of any one of claims 1 to 22, wherein the use of the multimeric ligand results in the number of alloreactive therapeutic cells being reduced, and wherein the therapeutic cells are T cells.

24. The use of claim 23, wherein the alloreactive T cells express CD3.

25. The use of any one of claims 1 to 24, wherein the use of the multimeric ligand results in the number of alloreactive therapeutic cells being reduced by about 90% or more.

26. The use of any one of claims 1 to 25, wherein after the use of the multimeric ligand, donor therapeutic cells survive in the subject that are able to expand and are reactive to viruses and fungi, wherein the therapeutic cells are T cells.

27. The use of any one of claims 1 to 25, wherein after the use of the multimeric ligand, donor T cells survive in the subject that are able to expand and are reactive to tumor cells in the subject.

28. The use of claim 1, wherein the at least one amino acid substitution is D330A.

29. The use of claim 1, wherein the at least one amino acid substitution is D330E.

30. The use of claim 1, wherein the at least one amino acid substitution is N405Q.

31. The use of any one of claims 1 to 30, wherein (a) the nucleic acid that encodes a chimeric polypeptide comprising the caspase-9 polypeptide or (b) the nucleic acid that encodes a chimeric polypeptide comprising the first caspase-9 polypeptide and the nucleic acid that encodes a chimeric polypeptide comprising the second caspase-9 polypeptide, comprises optimized codons encoding the caspase-9 polypeptide.

32. The use of any one of claims 1 to 31, wherein the modified caspase-9 polypeptide is encoded by the nucleic acid sequence of SEQ ID NO: 39 or is encoded by the nucleic acid sequence of modified caspase-9 polypeptide D330E of SEQ ID NO: 88.

33. The use of any one of claims 1 to 32, wherein the therapeutic cells are human cells.

34. The use of any one of claims 1 to 6, wherein the therapeutic cells are selected from the group consisting of mesenchymal stromal cells, embryonic stem cells, tumor infiltrating lymphocytes, and inducible pluripotent stem cells, progenitor cells, and hematopoietic progenitor cells.

35. The use of any one of claims 1 to 5, wherein the therapeutic cells are T cells.

36. The use of any one of claims 1 to 35, wherein the therapeutic cells are obtained or prepared from bone marrow, umbilical cord blood, peripheral blood, or peripheral blood mononuclear cells.

37. The use of any one of claims 1 to 36, wherein (a) the nucleic acid that encodes a chimeric polypeptide comprising the caspase-9 polypeptide or (b) the nucleic acid that encodes a chimeric polypeptide comprising the first caspase-9 polypeptide and the nucleic acid that encodes a chimeric polypeptide comprising the second caspase-9 polypeptide, is operably linked to a promoter.

38. The use of claim 37, wherein the chimeric polypeptide further comprises a marker polypeptide.

39. The use of claim 38, wherein the marker polypeptide is a ACD19 polypeptide.

40. The use of claim 38 or 39 further comprising a selection step, wherein cells that express the marker are selected for administration to the subject.

41. The use of any one of claims 1 to 40, wherein the caspase-9 polypeptide lacks the Caspase recruitment domain (CARD).

42. The use of any one of claims 1 to 41, wherein the multimerization region is selected from the group consisting of FKBP, cyclophilin receptor, steroid receptor, tetracycline receptor, heavy chain antibody subunit, light chain antibody subunit, single chain antibodies comprised of heavy and light chain variable regions in tandem separated by a flexible linker domain, and mutated sequences thereof.

43. The use of any one of claims 1 to 41, wherein the multimerization region is an FKBP12 region.

44. The use of claim 43, wherein the FKB12 region is an FKB12v 36 region.

45. The use of any one of claims 1 to 44, wherein the multimerization region is Fv'FvIs.

46. The use of any one of claims 1 to 45, wherein the multimeric ligand is selected from the group consisting of an FK506 dimer and a dimeric FK506 analog ligand.

47. The use of any one of claims 1 to 46, wherein the multimeric ligand is AP1903 or AP20187.

48. The use of any one of claims 1 to 47, wherein the multimerization region has an amino acid sequence of SEQ ID NO: 30, or a functional fragment thereof.

49. The use of claim 48, wherein the multimerization region having an amino acid sequence of SEQ ID NO: 30 is encoded by a nucleotide sequence in SEQ ID NO: 29, or a functional fragment thereof.

50. The use of claim 48 or 49, wherein the multimerization region further comprises a polypeptide having an amino acid sequence of SEQ ID NO: 32, or a functional fragment thereof.

51. The use of claim 50, wherein the polypeptide having an amino acid sequence of SEQ ID NO: 32, or a functional fragment thereof, is encoded by a nucleotide sequence in SEQ ID NO: 31, or a functional fragment thereof.

52. The use of any one of claims 1 to 51, wherein the therapeutic cells are transduced or transfected with a retroviral vector, an adenoviral vector, a lentiviral vector, or a gene expression vector.

53. The use of any one of claims 5 and 9, wherein a therapeutically effective level of therapeutic cells comprising the chimeric antigen receptor remain active in the subject following the use of the multimeric ligand.

54. The use of any one of claims 5, 9, and 53, wherein the chimeric antigen receptor is a chimeric T cell receptor.

55. The use of any one of claims 5, 9, and 53, wherein the chimeric antigen receptor comprises an scFv domain.

56. The use of any one of claims 1 to 55, wherein the subject is human.

57. The use of any one of claims 1 to 10, wherein the subject has graft vs. host disease and the use of the multimeric ligand alleviates the disease.

58. The use of claim 57, wherein the subject is human.

59. The use of any one of claims 1 to 58, wherein the subject exhibits symptoms of off-target or off-organ toxicity before the use of the multimeric ligand.

60. The use of any one of claims 1 to 59, wherein the subject exhibits symptoms of tumor lysis syndrome (TLS), cytokine release syndrome (CRS) or macrophage activation syndrome (MAS) before the use of the multimeric ligand.

61. The use of claim 60, wherein the use of the multimeric ligand alleviates the off-target or off- organ toxicity.

62. The use of claim 60, wherein the use of the multimeric ligand alleviates the tumor lysis syndrome (TLS), cytokine release syndrome (CRS) or macrophage activation syndrome (MAS).

63. The use of any one of claims 1 to 62, wherein the multimeric ligand is for administration to the subject in multiple doses, and at least 10% of the transplanted therapeutic cells that express the caspase-9 polypeptide or the modified caspase-9 polypeptide are killed following administration of a later dose of the multimeric ligand compared to the number of the transplanted cells before the later dose.

64. The use of any one of claims 1 to 62, wherein the multimeric ligand is for administration to the subject in multiple doses, with an escalation of dosage levels among the multiple doses.

65. The use of claim 64, wherein the escalation of dosage levels increases the number of therapeutic cells that are killed.

66. The use of any one of claims 1 to 65, wherein the multimeric ligand is for administration to the subject in a first dose and a second dose, wherein the second dose of the multimeric ligand is for administration more than 24 hours after the first dose of the multimeric ligand.

67. The use of claim 64, wherein the second dose of the multimeric ligand is for administration to the subject at least one week after the first dose of the multimeric ligand.