Expression of HIV inhibitors by mesenchymal stem cells

Abstract

A composition of matter and method of treating HIV with mesenchymal stem cells (MSC) is disclosed. Specifically, MSCs are transduced with vectors incorporating an anti-viral fusion inhibitor, such as a C46-derived peptide or neutralizing antibody. The transduced MSCs are capable of expression the inhibitor and preventing HIV virus-cell fusion.

Claims

1. A pharmaceutical composition, said composition comprising isolated mesenchymal stem cells (MSCs) wherein said isolated MSCs have been transfected with a lentiviral expression vector that is integrated into the genome of the isolated MSCs, wherein said lentiviral expression vector comprises a nucleic acid sequence encoding the full-length heavy and light chains of a single-chain antibody that is a human immunodeficiency virus (HIV) antiviral fusion inhibitor selected from the group consisting of N6, 10E8v4 and 35022, said lentiviral expression vector comprising a HIV-1 Ψ and, from 5′ to 3′, a Human cytomegalovirus (CMV) promoter, a nucleotide sequence encoding the heavy chain protein of the antibody, a nucleotide sequence encoding the light chain protein of said same antibody, and a P2A protease cleavage site separating said heavy and said light chain sequences, and conditioned media from the MSCs expressing the heavy and light chains inhibits HIV-1 infection.

2. The composition of claim 1, wherein said antiviral fusion inhibiting antibody is a neutralizing antibody.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Illustration of potential cell types upon MSC differentiation.

(2) FIG. 2 shows a diagram of the HIV fusion mechanism. From Drugs Fut. 1999, 24(12): 1355.

(3) FIG. 3 shows the design of a number of vectors.

(4) FIG. 4A shows the motility of MSCs in the presence of HIV-1 infection and FIG. 4B-C shows the migration of MSCs to towards e.g. HIV-1 released from chronically infected lymphocytes (ACH2 cells).

(5) FIG. 5. shows evidence that the MSCs inhibit HIV-1 infection and replication.

(6) FIG. 6 shows a determination of transduction level using GFP+ cells.

(7) FIG. 7 shows GFP+ levels by flow cytometry.

(8) FIG. 8 shows confirmation of a transduced RhBMS Differentiation Assay.

(9) FIG. 9A-B display western blots of MSCs transduced with various HIV peptide inhibitors packaged in virus vectors, wherein RhBMS cells are in 7A and 293T cells are in 7B.

(10) FIG. 10 displays a single round infection/inhibition assay in CEM×174 cells using conditioned media from MSC transduced with different inhibitors.

(11) FIG. 11A-C displays the design of a number of vectors incorporating antibody inhibitors. FIG. 11A shows an expression vector encoding the light chain of the N6 mAb; FIG. 11B shows an expression vector encoding the heavy chain of the N6 mAb;

(12) FIG. 11C shows a lentiviral vector encoding both the light and heavy chains of the N6 mAb with a P2A self-cleaving element to separate the heavy and light chains after expression.

(13) FIG. 12 displays varying concentrations (10%, 25%, and 50%) of conditioned media expressing B404 against SIV to inhibit viral infection of TZM-bl reporter cells.

(14) FIG. 13 displays varying concentrations (10%, 25%, and 50%) of conditioned media expressing N6, 10E8v4, and/or 35022 against HIV-BaL to inhibit viral infection of TZM-bl reporter cells.

DETAILED DESCRIPTION

(15) The disclosure describes novel transduced mesenchymal stems cells (MSC) that express HIV virus-cell fusion inhibitors and a method of use to treat HIV. Specifically, mesenchymal stems cells (MSC) are transduced with one or more retroviral and/or lentiviral vectors expressing antiviral fusion inhibitors of HIV is disclosed. The transduced MSCs express the inhibitors without losing important MSC characteristics such as the ability to differentiate.

(16) Methods of using the novel composition can comprise treating an animal, and preferably a human, suffering from an HIV infection wherein a peptide inhibitor or neutralizing antibody is incorporated into a viral or nonviral vector, which is then transduced into a MSC such that the MSC can express the inhibitor. The transduced MSCs are then administered to a subject alone or in combination with anti-retroviral therapies. The MSCs can be autologous or allogenic and taken from any source including bone marrow or adipose tissue. Preferred viral vectors are lentivirus, retrovirus, or any combination thereof. Exemplary inhibitors include one or more of secreted antiviral entry inhibitory (SAVE) peptides, C46, maC46 or neutralizing antibodies. The MSCs can be administered intravenously, subcutaneously, or intraperitonealy.

(17) FIG. 2 displays the fusion mechanism by which HIV infects new cells. HIV fusion with a target cell starts with the attachment of gp120 to the N-terminal domain of the CD4+ receptors (B) Once attached, gp120 undergoes a conformational change that unmasks the binding site for the cell co-receptor (C). Once the virus is bound, a conformational change exposes the N-terminal hydrophobic region of gp41 to the CD4+ cell membrane and becomes the “pre-hairpin intermediate”. Gp41 undergoes a conformation change (D), also referred to as a coiled-coil bundle, to bring the virus and cell membranes together, mediating fusion of virus and cell (E).

(18) In one aspect of the present disclosure, to inhibit the fusion of HIV, MSCs are transduced with HIV peptide inhibitors. Many HIV peptide inhibitors exist including maC46 and C46 peptides. The HIV entry inhibitor maC46 is a membrane-anchored peptide derived from the second heptad repeat of the HIV-1 transmembrane glycoprotein gp41. C46 peptides are members of the new fusion inhibitor class of antiretroviral drugs that efficiently block infection of new cells by interfering with the function of gp41. The C46 peptides can be membrane-bound or secreted.

(19) The peptide inhibitors are incorporated into retroviral or lentiviral vectors, which act as gene delivery vehicles for transducing the MSCs with the peptide inhibitors. Exemplary vectors include murine leukemia (MLV) and self-inactivating lentiviral vectors (HSRT), however other viral or non-viral vectors systems would also be applicable. FIG. 3 displays a few of the virus vectors that have incorporated the peptide inhibitors, particularly the maC46 and SAVE peptide sequences. The vectors can also contain a fluorescence marker, such as GFP, to aid in monitoring the success of the transduction.

(20) Similarly to the peptide inhibitors, MSCs can be transduced with HIV antibody inhibitors to inhibit the fusion of HIV. Many HIV antibody inhibitors, also referred to as antiviral fusion inhibiting antibody herein, exist, including neutralizing antibodies. Broadly neutralizing antibodies (bNAbs), for example, can be used to target HIV epitopes, thus neutralizing multiple HIV strains, even if the virus mutates.

(21) After being transduced, the MSC can be analyzed to confirm expression of the peptide inhibitors using fluorescent microscopy, PCR, differentiation assays and other commonly used techniques.

(22) The present disclosure is exemplified with respect to the examples and figures below. However, this is exemplary only, and the disclosure can be broadly applied to incorporate any HIV fusion inhibitor for all phases of the HIV life cycle and can be used for any animal. The following experiments and examples are intended to be illustrative only, and not unduly limit the scope of the appended claims.

Example 1

(23) The overall idea of the presently disclosed method is to use MSCs with modified genomes as a ‘Trojan horse’ bioreactor to treat HIV. MSCs are known to migrate to sites of infection and tumors. However, MSCs have never been shown to migrate to virus-infective cells. Such migration is of particular importance for the presently disclosed methods, as the MSCs will be used to carry and express a large variety of HIV inhibitors at the sites of virus-infected cells.

(24) For the first time, evidence of MSC migration to local, virus-infective sites is being show. FIG. 4A shows the motility of MSCs in the presence of an HIV-1 infection. As seen in FIG. 4B-C, the MSCs migrate towards HIV-1 released from chronically infected lymphocytes (ACH2 cells). In FIG. 5, the MSCs are inhibiting the HIV-1 infection and replication process.

(25) Results:

(26) MSCs have been proven to be capable of migrating to cells infected with a virus. However, long term, continuous expression of inhibitors is desired as a proper treatment method for the virus. Thus, MSCs that can continuously express multiple inhibitors to treat HIV viruses, regardless of the location of the infected cells, are a desirable treatment method.

Example 2

(27) Gene transfer and expression was performed by the following steps:

(28) Cells:

(29) Human Bone Marrow stem cells (HuMSCs), Rhesus Bone Marrow Stem cells (RhMSCs), and Rhesus Adipose stem cells (RhACSs) were plated at 10,000/cm.sup.2 in alpha-MEM/20% FBS with no antibiotics the day before transduction.

(30) For transduction, the plated cells were exposed to various MOI of the control vectors, such as LZRS-GFP (19) or HRST-cmvGFP (38), or the therapeutic vectors, such as T42 (unknown), T60 (22), and M218 (unknown), containing the C46 peptide sequences shown in FIG. 3. Then, the plates were centrifuged at 1000 rpm for 1-2 hours. After which, the MSCs were cultured at 37° C. and 5% CO.sub.2 for two days. After Day 2, cells were returned to standard culture conditions, wherein the cells were re-suspended in complete conditioned media and filter through a 70 mm cell strainer.

(31) Expression Confirmation:

(32) Fluorescent microscopy and flow cytometry were used to assess the expression of the C46 peptide using GFP. Real time PCR was also performed.

(33) As shown in FIG. 6, cells were observed under fluorescent microscope to confirm transduction and expression of GFP in transduced cells. The upper panels are, from left, non-transduced (NT) MSC, rhASC with phase contrast, and GFP expression overlayed onto the phase contract image. Lower panels show overlay of GFP expression in HuMSCs, RhMSCs, and RhACSs transduced with HRST-cmvGFP. As seen, the GFP is readily expressed in the human and rhesus stem cells.

(34) FIG. 7 displays the flow cytometry Fluorescence-activated cell sorting (FACs) results for HuMSCs, RhMSCs, and RhACSs. For this analysis, cells were trypsinized and washed with 1% mouse serum. After the wash, cells were fixed with 4% paraformaldehyde and analyzed for GFP expression by flow cytometry. The NT control population was set as background fluorescence.

(35) The HRST-GFP showed the highest expression levels in all three populations by FACs analysis. The LZRS-transduced MSC showed slightly elevated levels of GFP expression in all three MSCs populations.

(36) Gene transfer and expression was detectable up to 69% in MSCs by fluorescent microcopy and flow cytometry.

(37) The real time PCR results for the vector backbone, shown below, detected between 5-25% of the human MSCs, rhesus MSCs, and HEK 293 cells contained the Psi packaging element in T60 and M218. Both T60 and M218 are murine leukemia virus based vectors.

(38) TABLE-US-00002 C46 Vector Cell type T60 (%) M218 (%) Hu MSC 3.9 6.3 Rh MSC 12.5 25.5 Rhs ASC 10.6 14.7 293T 10.0 13.3

(39) Differention assays were also performed to confirm transduced RhMSCs to test that the inhibitors do not disturb MSCs differentiation and results are shown in FIG. 8. The osteogenic, adipogenic, and chrondrogenic differentiation abilities of the MSCs were taken at 21 days for the M218, T60, HRST-GFP and T42 vector. As seen in FIG. 8, the MSCs retained their ability to differentiate into multiple cells, regardless of the vector or peptide inhibitor used. This shows that MSCs have not lost their differentiation abilities after being transduced.

(40) Results:

(41) The HIV1-based lentiviral vector HRST-cmvGFP was superior to LZRS-GFP in both the frequency of gene transfer and the level of expression. Furthermore, real-time PCR data showed a transduction rate of 5-25% in the MSCs for MLV C46 vectors.

Example 3

(42) The above method in Example 2 was applied to a larger variety of cells.

(43) Cells:

(44) Most cells were obtained from the NIH AIDS Reagent Program. The NIH AIDS Reagent Program is a worldwide resource for state-of the art HIV research materials, some of which are not yet commercially available. CEM×174 cells (NIH AIDS Reagent Program Cat no. 272), expressing CD4, T and B cell markers, were obtained because they are easily infected with HIV virus. Second T-cell line “PM-1” (NIH AIDS Reagent Program Cat no. 3038), permissive for growth of macrophage and T cell tropic viruses, a subclone of HuT78 (Human T cell lymphoma) expressing CD4, CXCR4, and CCR5, was obtained. All T-cell lines were cultured in Roswell Park Memorial Institute medium (RPMI) 1640 medium.

(45) Human TZM-bl cell (NIH AIDS Reagent Program Cat no. 8129), previously designated JC53-bl (clone 13) is a HeLa cell line. The parental cell line (JC.53) stably expresses large amounts of CD4 and CCR5. The TZM-bl cell line was generated from JC.53 cells by introducing separate integrated copies of the luciferase and ß-galactosidase genes under control of the HIV-1 promoter. All media were supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 10,000 U/mL penicillin/streptomycin.

(46) Rhesus macaque bone marrow/adipocyte derived mesenchymal stem cells were isolated and maintained in complete culture medium, 20% FBS MEM α (Minimum Essential Medium α), with 1-glutamine, fetal bovine serum (Atlanta Biologicals Premium Select FBS), Penicillin G (10,000 units/mL), and streptomycin sulfate (10,000 μg/mL) in solution of 0.85% NaCl (Invitrogen/GIBCO).

(47) Human embryonic kidney cell line 293T was obtained from the ATCC, and was maintained in Dulbecco's modified Eagle's medium (DMEM).

(48) Bone Marrow and Adipose Derived Mesenchymal Stem Cells Isolation and Culture:

(49) Bone marrow-derived mesenchymal stem cells were isolated by discontinuous density gradient centrifugation following the Tulane Center for Stem Cell Research and Regenerative Medicine protocol. Briefly, LSM (Lymphocyte Separation Medium, ICN50494, MP Biomedicals, No.: 0850494) one-step separation medium was underlayered beneath the bone marrow mixture. The sample was centrifuged at 1400 rpm (˜400 g) for 40 min. The low-density mononuclear cells were collect at the white interface and washed with fresh media. After the last centrifugation, the supernatant discard and a lysis buffer (ACK lysing buffer, Gibco, catalog no. 10492-01) was used to eliminate residual red blood cells.

(50) The collected cells were plated at a concentration of ˜1-2 million cells in a 15 cm tissue culture dish and incubate at 37° C. in 5% CO.sub.2. The complete culture medium was changed the next day (24 hours later) and replace with fresh complete medium. When the plate was about 70-80% confluent (˜14 days), cells were trypsinized and frozen at P0 in Liquid N.sub.2 until they were expand to P1 or frozen as a pellet for DNA and protein analysis.

(51) Adipose-derived stem cells were isolated according to the Tulane Center for Stem Cell Research and Regenerative Medicine protocol. Briefly, the adipose tissue was washed extensively with a 5% Phosphate Buffer Solution (PBS 5% p/s) before being minced into small pieces using a sterile scalpel. The pieces were placed in a 15 cm dish with 0.075% Collagenase Type I (Gibco, catalog no. 17100-017) in PBS. Collagenased tissues were incubated for 30 min at 37° C. at 5% CO.sub.2. The liquid portion was removed and the plate was washed several times with PBS 2% p/s, and centrifuge to obtain a pellet. After lysing with ACK Lysing Buffer (Gibco, catalog no. 10492-01), the adipose-derived cells were washed and incubated for 10 min on ice to lyse any red cells. The cells were again washed with 20 ml PBS 2% p/s, wherein the supernatant was discard. The cell pellets were re-suspended in complete conditioned media and filter through a 70 mm cell strainer.

(52) Collected cells were plated into the proper size culture dish depending on the size of cell pellet and incubate at 37° C. at 5% CO.sub.2. The media was changed the next day and replaced with fresh media. When the plate was about 70-80% confluent, aspirate the medium. Cells were frozen and stored in liquid N.sub.2 until they used to expand to P1, and then were frozen a pellet for DNA and protein analysis.

(53) Peptide Cloning and Mesenchymal Stem Cells Transduction:

(54) The C46 peptide used in the present experiment was SEQ No. 1.

(55) C46 peptides driven by Murine leukemia virus (MLV) and lentivirus (M218, T60, and T42) and control vectors (LZRS-GFP and HRST-cmv-GFP) were transformed using one shot stb13 kit with competent cells (Invitrogen, Cat. No. C7373-03) followed by manufacture's procedure. After sitting overnight, the colonies were selected and a single colony was grown in LB medium plus ampicillin overnight. DNA mini prep was performed with PureLink® Genomic DNA Mini Kit (Invitrogen, Catalog No. 1820-01).

(56) Transient transduction of 293T cells with MLV and lentiviral vectors were completed by the calcium phosphate precipitation method. The calcium phosphate transduction method is used for introducing DNA into mammalian cells and is based on forming a calcium phosphate-DNA precipitate. Calcium phosphate facilitates the binding of the DNA to the cell surface. DNA then enters the cell by endocytosis.

(57) 15 μg of MLV vector plasmid was used for LZRS-GFP and LZRS-T60 and 2 ug of LZRS-M218 was used for transfection of the Phoenix (amphotropic) packaging cell line, a cell line bases on HEK 293T cells. They constitutively express the MLV Gag/Pol and amphotropic envelope genes. Addition of the vector genome will generate replication defective MLV-based retroviral particles in the supernatant. For the HIV-based lentiviral vectors, 13 ug of HRST-GFP or T42 vector DNA was transfected along with the HIV packaging plasmids [pHDM-Hgm2 (gag/pol), pRC/Rev, pHDM-Tat 1b, envelope HIV-1 JRFL or VSV-G (pHDM.G)] or packaging vector (M438).

(58) Cell culture supernatants containing replication defective lentiviral particles were collected from 24 to 48 hours post-transduction, filtered (0.22 μm pore size) and stored at −80° C. until use.

(59) Transduction of Mesenchymal Stem Cells with Lentiviral and MLV Vectors:

(60) 1×10.sup.5 cells/well of MSCs were plated in 6 well-plate a day before transduction. A desired amount of viral supernatant was added into each well, such as 1 ml of non-concentrated viral supernatant or 100 ul of concentrated viral supernatant. MSCs were transduced with MLV vector and lentiviral vector supernatants by spinoculation (1 hr, 1200 rpm, 31° C.) then incubated at 37° C. in 5% CO.sub.2.

(61) At day 2, the viral supernatant was removed and fresh 20% FBS alpha-MEM with no antibiotics was added to the MSCs. This media was change with 20% FBS alpha-MEM with no antibiotics at day 4. At day 6-7, depending on cell density, cells were prepared for fluorescent microscopy, flow cytometry, and quantitative proviral integrations by real-time PCR. Transduction efficacies were determined 3 days after transduction by flow cytometry.

(62) Detection of Secreted Version of C Peptides:

(63) To determine the secreted C46 peptides in mesenchymal cell culture supernatants and cell lysates, a western blot analysis was performed and is shown in FIGS. 9A and B. Human HIV-1 gp41 monoclonal antibody (2F5) was obtained from the NIH AIDS Reagent Program. 2F5 allowed the binding of the antibody to all available C peptides.

(64) The secreted peptides were apparent at the 5.82 kD level. This shows that the peptide inhibitors are being secreted by the MSCs and can be available for HIV treatments.

(65) Preparation of HIV Pseudotyped Particles:

(66) Day before transduction, 9×10.sup.6 cells of 293T cells were plated on 15 cm culture dish for a total of 10 plates. Additional plates were prepared for VSV-G control. 80% of confluency was confirmed under microscopy on the day of transduction.

(67) For the transient transfection, HRST-GFP was mixed with pHDM-Hgm2 (gag/pol), pRC/Rev, pHDM-Tat 1b, and envelope gene (either HIV-1 JRFL envelope or the VSV-G control envelope, pHDM.G). All the listed plasmid vectors were gift from Dr. Richard C. Mulligan, Harvard Gene Therapy Institute except JRFL envelope plasmid was provided by Dr. Dorothee von Laer from Innsbruck Medical University. 2.5M CaCl.sub.2 and 2×HBS solution was added equal ratio. Final volume of 1 ml of transduction mixture was applied into each culture dish. After 6 hours later, fresh 10% DMEM medium was replaced. Viral supernatant was collected 24-48 hrs after transduction and filtered through 0.22 um size and treated with 5×PEG-it virus precipitation solution (PEG-It™ Virus Precipitation Solution (5×), SBI, Cat. #LV810A-1/LV825A-1) for 48-72 hrs. Yellow beige colored pellets were confirmed and, following manufacture's protocol, the virus was concentrated by centrifuge at 1500 g for 30 min at 4° C. Virus pellets were collected and resuspended in PBS solution and stored at −80° C. until used.

(68) Viral Single Round Infection/Inhibition Assay of Pseduotyped HIV-1 System by C46 Transduced in MSCs:

(69) In order to mimic the HIV infection system, HRST (CMV-GFP) was packaged with an HIV envelope, JRFL. The CD4+ cell line CEM×174 were treated with conditioned medium (CM) from the control (HRST-GFP, LZRS-GFP) or C46 (T-60, M218, and T-42) transduced MSC and infected with various MOI of concentrated pseudo HIV-1 viral-like particles produced by calcium phosphate method. After 48 hrs, cells were harvested and fixed with 4% PFA. Samples were analyzed for GFP(+) by BD FAC Verse™ flow cytometry at Tulane National Primate Research Center. GFP (+) flow analysis was conducted by FlowJo version 10 program.

(70) The resulting single round infection/inhibition assay is shown in FIG. 10. The CM from the control cells had the highest level of GFP expression (infection with the pseudoviral particles). Treatment with CM from the C46-transduced MSCs blocked infection. The level of GFP expression in the CEM×174 cells was graphed relative to the expression in the control population. Thus, the conditioned media from C46-transduced MSCs were able to block infection with HIV-1 pseudovirus in a single round inhibition assay.

(71) Mesenchymal Stem Cell Differentiation Assay:

(72) To test that the inhibitors do not disturb MSCs differentiation, osteogenic, adipogenic, and chondrogenic differentiation assay with StemPro® Osteogenesis Differentiation Kit, Gibco A1007201, A1007001, and A1007101 was performed on LZRS-GFP, LZRS-T60, HRST-GFP, M218, T42, and non-transduced RhMSCs. Assay procedure was followed manufacture's protocol. Briefly, cell number to be seeded for assay was 5×10.sup.3 cells/cm.sup.2 and 1×10.sup.4 cells/cm.sup.2 into 12 wells for osteogenic and adipogenic differentiation respectively. 1.6×10.sup.7 viable cells/ml was plated by seeding 5-μl droplets of cell solution in the center of multi-well plate wells for classical stain for chondrogenic differentiation. Following around 21 days culture depends on cells growth, osteogenic, adipogenic, and chondrogenic differentiation were stained by alizarin red S, oil red o, and safranin o solution respectively. Stained cells were observed and taken pictures under bright field light microscope.

Example 4

(73) The MSCs that expressed HIV inhibitor peptides in Examples 2 and 3 can be combined with antiviral drugs in vitro to assess the synergetic effects. PMPA (Tenofovir disoproxil fumarate, TDF or tenofovir) and FTC (Emtriva or Emtricitabine) are exemplary antiviral drugs whose synergetic effects with the above MSCs can be measured in an in vitro cell culture model.

(74) For instance, CEM×174 cells can be infected with various MOI of Simian-HIV 89.6p or HIV strains. Then, MSCs can be treated with Mitomycin C, which is used to generate mitotically inactive the MSC feeder cells. The CEM×174 cells can then be co-cultured with the treated MSC for up to 14 days with or without additional antiviral drugs (e.g. 2 uM of PMPA and 1.5 uM FTC or others). The HIV replication in the CEM×174 cells can be monitored using p27 flow cytometry or ELISA, or RT-PCR, or other method. Inhibition of viral replication will be quantified by limiting dilution assay.

(75) Alternatively, CEM×174 cells can be treated with conditioned medium from the control (HRST-GFP, LZRS-GFP) or C46 (T-60, M218, or T-42) transduced MSCs and combined with the PMPA and FTC (or other standard HIV drug). Then, CEM×174 cells are exposed to the HIV viral-like particles. Inhibition of the HIV infection of the CEM×174 cells is measured by GFP expression.

(76) Additionally, the order of treatment can be varied such that infected CEM×174 cells are treated with the transduced MSCs before the antiviral drugs or co-administered with the antiviral drugs.

Example 5

(77) Animal model testing using the MSCs that expressed HIV inhibitor peptides in Examples 2 and 3 alone or in combination with antiviral drugs can be performed with small and large animal models.

(78) An exemplary small animal model is an immunodeficient mouse model. Table 1 lists many available mouse models and their advantages or disadvantages indicating that certain mouse models may be better candidates for animal testing.

(79) TABLE-US-00003 TABLE 1 Exemplary immunodeficient mouse model Cellular Humanized composition in Mouse reconstituted hu- Model mice Advantages Disadvantages Hu-PBL T and B cells Easy to generate No multilineage SCID Can immediately be used haematopoiesis after transfer of PBLs Limited time fram for experiments Strong activation of T-cells Emergence of xeno-reactive T-cells (GVHD) Thy/Liv T cells Organoid of fetal Surgical skills needed SCID hu Single positive, thymus/liver tissue with Human fetal tissue needed double positive sustained T-cell No multilineage and double lymphopoiesis hematopoiesis negative Valuable to study certain Lack of CCR5 expression on Thymocytes pathogenic aspects intrathymic T progenitor cells Rag2 T, B cells, Long term multilineage Delay between transplantation of .sup.−/−   .sup.−/− Monocytes, hematopoiesis human CD34+ cells and Macrophages, NK Specific antibody development of lymphoid cells, and DCs response to recall systems of ~15 weeks antigens Suited to study HIV pathogenesis, HIV latency, gene therapy and novel anti-HIV treatment approaches NOG or T, B cells, Higher reconstitution Sensitive to irradiation NSG Monocytes, levels as compared to Macrophages, NK Rag mice cells, and DCs Suited to study HIV pathogenesis, HIV treatment, gene therapy approaches NOD/SCID- T, B cells, Generation of adaptive Two stop procedure for hu BLT & Monocytes, immune responses generating BLT mice NOD/SCID Macrophages, NK Suited to study HIV Surgical skills needed    .sup.−/− (NSG) cells, and DCs pathogenesis, HIV Human fetal tissue needed BLT latency, gene therapy and novel anti-HIV treatment approaches

(80) The therapeutic potential of the stem cells in the humanized mouse models can be measured by analyzing the degree of human cell engraftment by flow cytometry of the peripheral blood mononuclear cells after they have been stained for panhuman markers such as CD45, CD3, CD4, CD8, CD14, and CD19.

(81) Non-human primate models can be used for the large animal model testing. Such primate models would allow for the use of simian-HIV (S-HIV) infections. The S-HIV viral load in plasma of the animals can be measured using real time PCR. The therapeutic potential of the stem cells can be determined by analyzing the blood and tissue samples from infected primates for bio-distribution of MSCs, toxicity and peptide measurement.

Example 6

(82) In addition to peptide inhibitors, antibody-based inhibitors can also be incorporated into the MSCs genome for long-term expression and treatment of virus-infected cells. Similar to the peptides, a variety of neutralizing antibodies were incorporated into vectors and expressed in cells. For the experiments involving antibody-based inhibitors, transient transfection of two expression vectors (plasmids encoding N6 mAb heavy and light chains) were used, each using a CMV early promoter to regulate expression (see vector maps in FIG. 11A-B). Alternatively, a lentiviral vector could have also been used to deliver both the N6 mAb H&L genes with a P2A self-cleaving element to separate the heavy and light chain proteins after expression (see FIRST map in FIG. 11C), much like it was used above with the protein inhibitors. Conditioned media from the transfected MSCs was used to inhibit HIV-1 viral replication. The effects for multiple NABs (B404, N6, 10E8v4, and/or 35022) are shown in FIGS. 12-13.

(83) FIG. 12 shows viral-specific activation of the luciferase gene in TZM-bl cells. The TZM-bl cells (2×10.sup.4 cells/well) and SIVmac239 were pretreated with CM (50%, 25%, or none) for 2 hours at 37° C., and then mixed. After 72 hours, the cells were washed with PBS before being lysed with 1× passive lysis buffer. Firefly luciferase activity was then measured from clear lysates, as a percent of control. Cells challenged with SIV produce high levels of luciferase activity. However, when the cells were treated with CM containing B404 Nab, viral replication was significantly inhibited.

(84) FIG. 13 displays varying concentrations (10%, 25%, and 50%) of conditioned media expressing N6, 10E8v4, and/or 35022 to inhibit viral infection of TZM-bl reporter cells. The TZM-bl cells were prepared as described above. The TZM-bl cells (2×10.sup.4 cells/well) and HIV-BaL (MOI=5) were treated with dilutions of NAB (10%, 25%, and 50%) for 2 hours at 37° C., and then mixed. After 72 hours, the cells were washed with PBS before being lysed with 1× passive lysis buffer. Firefly luciferase activity was then measured from clear lysates as percent of control.

(85) As expected, CM containing NABs was able to inhibit the HIV infection in the TZM-bl reporter cells for both SIV and HIV-BaL. The inhibition data is very promising. The control cells maintained low levels of enzyme, but infection with HIV-BaL produce high levels of Luc activity, and pretreatment with the NAB, even when diluted to only 10% CM, potently inhibited viral replication. For example, with N6, Applicant was able to inhibit 99% of HIV infection with only 10% mixture of conditioned media. Other types of antibodies, or even combinations of antibodies, are expected to show similar success with the treatment of cells infected with HIV. This data shows that HIV can be treated using less complex and more accommodating methods that rely on MSCs expression of peptides and/or antibodies.

(86) In view of the above experiments, it is clear that MSCs can carry a variety of genes for viral inhibitors to the local site of inflammation or virus infective cells. These inhibitors can be incorporated into many types of expression vectors, such as retroviral or lentiviral vectors, which are then transduced into the MSCs for expression at the site of inflammation and/or infection.

(87) While certain novel features of this disclosure shown and described above are pointed out in the annexed claims, the disclosure is not intended to be limited to the details specified, since a person of ordinary skill in the relevant art will understand that various omissions, modifications, substitutions and changes in the forms and details of the disclosure illustrated and in its operation may be made without departing in any way from the spirit of the present disclosure. No feature of the disclosure is critical or essential unless it is expressly stated as being “critical” or “essential.”

(88) Furthermore, the above experiments and examples are not intended to limit the scope of the disclosure composition or methods to certain cells or animals.

(89) The following references are incorporated by reference in their entirety. WO2010053350 US20100226976 US20110027240