GENETICALLY MODIFIED MESENCHYMAL STEM CELLS EXPRESSING ALPHA-1 ANTITRYPSIN (AAT)

Abstract

Genetically modified mesenchymal stem cells can be used as a medicament in the treatment of medical conditions associated with inflammation and/or an unwanted immune response in subjects without an alpha1-antitrypsin (AAT) deficiency. The stem cells include an exogenous nucleic acid, which includes (i) an Alpha-1 antitrypsin (AAT) encoding region operably linked to (ii) a promoter or promoter/enhancer combination.

Claims

1. A method for treating a subject having a medical conditions associated with inflammation and/or an unwanted immune response without an alpha1-antitrypsin (AAT) deficiency, wherein the method comprises administering genetically modified mesenchymal stem cells to the subject, wherein said genetically modified mesenchymal stem cells comprise an exogenous nucleic acid comprising (i) an Alpha-1 antitrypsin (AAT) encoding region operably linked to (ii) a promoter or promoter/enhancer combination.

2. The method according to claim 1, wherein the exogenous nucleic acid comprises a viral vector.

3. The method according to claim 2, wherein the viral vector is a retroviral vector.

4. The method according to claim 1, wherein the promoter or promoter/enhancer combination is a constitutive promoter.

5. The method according to claim 4, wherein the constitutive promoter is the EFS, PGK, or EF1alpha promoter.

6. The method according to claim 1, wherein said promoter or promoter/enhancer combination is an inducible promoter.

7. The method according to claim 6, wherein the promoter is inducible upon differentiation of said cell post-administration.

8. The method according to claim 6, wherein the promoter is an inflammation-specific promoter.

9. The method according to claim 6, wherein the promoter is the Tie2, HSP70 or RANTES promoter.

10. (canceled)

11. (canceled)

12. The method according to claim 1, wherein a therapeutically effective number of genetically modified mesenchymal stem cells according to claim 1 are introduced into the bloodstream of the subject.

13. The method according to claim 12, wherein the medical condition associated with inflammation and/or an unwanted immune response is a lung disease.

14. The method according to claim 13, wherein said lung disease is a respiratory disease.

15. The method according to claim 13, wherein the lung disease is acute lung injury, chronic obstructive pulmonary disease (COPD) including chronic bronchitis, emphysema, bronchiectasis and bronchiolitis, acute respiratory distress syndrome, asthma, sarcoidosis, hypersensitivity pneumonitis and/or pulmonary fibrosis.

16. The method according to claim 13, wherein said therapeutically effective number of genetically modified cells are introduced to the lung of the subject by inhalation, optionally in combination with introduction of said cells into the bloodstream of the subject.

17. The method according to claim 12, wherein the medical condition associated with inflammation and/or an unwanted immune response is gout.

18. The method according to claim 1, wherein the medical condition associated with inflammation and/or an unwanted immune response is chronic fibrosis.

19. The method according to claim 12, wherein the inflammatory disease is of the kidney, liver and/or colon of the subject.

20. The method according to claim 12, wherein the medical condition associated with inflammation and/or an unwanted immune response is an inflammatory disease selected from the group consisting of vasculitis, nephritis, inflammatory bowel disease, rheumatoid arthritis and/or Graft versus Host disease.

21. The method according to claim 12, wherein the medical condition associated with inflammation and/or an unwanted immune response is an autoimmune disease.

22. The method according to claim 21, wherein the autoimmune disease is diabetes Type 1.

23. The method according to claim 12, wherein the therapeutically effective number of genetically modified mesenchymal stem cells is introduced into the bloodstream of the subject via intravenous injection.

24. The method according to claim 13, wherein the lung disease is an inflammatory disease of the lung.

25. The method according to claim 18, wherein the chronic fibrosis is of the kidney, liver and/or colon of the subject.

Description

FIGURES

[0116] The following figures are presented in order to describe particular embodiments of the invention, by demonstrating a practical implementation of the invention, without being limiting to the scope of the invention or the concepts described herein.

Short Description of the Figures

[0117] FIG. 1: Preferred expression cassettes of the present invention.

[0118] FIG. 2: Titration of retroviral supernatants on HT1080 cells.

[0119] FIG. 3: Intracellular flow cytometry analysis of primary human MSCs transduced with viral expression constructs.

[0120] FIG. 4: Assessment of transgenic AAT expression by ELISA.

[0121] FIG. 5: Inhibition of Neutrophil Elastase by AAT expressed from transduced MSCs.

[0122] FIG. 6: Experimental design BLM-induced lung fibrosis model

DETAILED DESCRIPTION OF THE FIGURES

[0123] FIG. 1: Preferred expression cassettes of the present invention.

[0124] Schematic representation of the preferred expression cassettes of the present invention. Numbers depicted in the figure represent apceth's internal plasmid denomination and will be used herein for reasons of simplification. The promoters are shown by a lower case p. LTR elements relate to long terminal repeats of the gamma retroviral vector employed. An internal ribosome entry site is abbreviated as IRES. A Posttranscriptional Regulatory Element is abbreviated as oPRE. A puromycin resistance gene (pac) is employed for selection.

[0125] FIG. 2: Titration of retroviral supernatants on HT1080 cells.

[0126] Larger constructs (e.g. 161 and 164 in which the SERPINA1 cDNA is driven by the full length EF1a promoter) yield reduced although sufficient titers as compared to smaller constructs such as ones comprising the EFS promoter (e.g. 159 or 194). In the present experiment, the highest titer is obtained with the lentiviral construct 215.

[0127] FIG. 3: Intracellular flow cytometry analysis of cells transduced with viral expression constructs.

[0128] FIG. 3a: Intracellular flow cytometry analysis of primary human MSCs transduced with viral expression constructs—% ic AAT positive cells. FIG. 3a depicts percentage of MSCs that stained positive for intracellular AAT after transduction with different gamma-retroviral and lentiviral expression constructs. FIG. 3b: Intracellular flow cytometry analysis of primary human MSCs transduced with viral expression constructs—mean fluorescence intensity (MFI). FIG. 3b depicts mean fluorescence intensity (MFI) values of MSCs transduced with different gamma-retroviral and lentiviral expression constructs. All tested vector constructs are able to transduce primary human MSCs, and transgenic AAT is detected by intracellular flow cytometry in all transduced samples. Differences in transduction efficiency as measured by % is AAT positive cells are most likely due to different starting titers of the viral supernatants used for transduction. Different expression levels as analyzed by MFI are the result of distinct promoters utilized as well as varied gene cassette constellations.

[0129] FIG. 4: Assessment of transgenic AAT expression by ELISA.

[0130] Transgenic AAT expression in primary human MSCs is confirmed in all samples by ELISA. Variances in the amounts expressed are due to distinct promoters utilized in the vectors as well as varied gene cassette constellations.

[0131] FIG. 5: Inhibition of Neutrophil Elastase by AAT expressed from transduced MSCs.

[0132] AAT expressed from transduced primary human MSCs is functional and inhibits Neutrophil Elastase at levels comparable to medium containing 10% serum or ˜1.5 μM SPCK.

[0133] FIG. 6: Experimental design BLM-induced lung fibrosis model (adapted from Tashiro et al., 2015).

Examples

[0134] The invention is further described by the following examples. These are not intended to limit the scope of the invention. The experimental examples relate to the development of technology that enables Alpha-1 antitrypsin (AAT) expression from genetically modified MSCs. The examples further relate to therapeutic trials encompassing the treatment of conditions of the lung.

[0135] In preferred embodiments the examples relate to the preclinical development of a novel gene therapy product that combines the anti-inflammatory effect of Alpha-1 antitrypsin (AAT) with the immunomodulatory properties of primary human mesenchymal stem cells (MSCs) for the treatment of inflammatory lung diseases.

[0136] 1. Design and Cloning of Retroviral Vector Constructs:

[0137] The transgene expression cassettes are constructed using standard cloning techniques as described in Julia Lodge, Peter Lund, Steve Minchin (2007) Gene Cloning, New York: Tylor and Francis Group. The gene expressed by these constructs is the human SERPINA1 cDNA {Homo sapiens serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 (SERPINA1), transcript variant 1, mRNA; NCBI Reference Sequence: NM_000295.4, encoding Alpha-1 antitrypsin (AAT)}. A codon optimized cDNA as described above according to SEQ ID NO 2 has also been assessed.

[0138] The SERPINA1 gene as described herein is expressed by activation of different constitutive promoters, such as the human EEF1A1 eukaryotic translation elongation factor 1 alpha 1 promoter (pEF1a), the short form of the human EEF1A1 eukaryotic translation elongation factor 1 alpha 1 promoter (pEFS), or the human phosphoglycerate kinase promoter (pPGK). The promoters may also be inducible promoters like Tie2, RANTES or the HSP70 promoter. The gene may or may not be fused with tag-sequences (e.g. marker proteins/peptides such as the hemagglutinin tag or the HIS tag) to allow for easy detection of expression later on (Hinrik Garoff, 1985, Annual Review of Cell Biology, Vol. 1: 403-445).

[0139] The expression cassette may or may not include a second transgene cassette consisting of a selectable marker gene, such as a cell surface marker or a resistance gene (for example the pac gene to confer puromycin resistance) to allow for enrichment of genetically modified cells later in the process (David P. Clark, Nanette J. Pazdernik, 2009, Biotechnology: Applying the Genetic Revolution, London: Elsevier). The gene is either driven by a separate promoter or located 3′ of an IRES sequence within the SERPINA1 expression cassette.

[0140] To assess potential positional effects, SERPINA1 and pac cassettes are cloned in different constellations (SERPINA1 cassette 5′ of pac cassette and vice versa). Refer to FIG. 1.

[0141] The expression cassettes disclosed in FIG. 1 are then inserted into a suitable vector system, e.g. a gamma-retroviral (e.g. pSERS11, EP2019134A1) or lentiviral backbone (e.g. U.S. Pat. No. 8,846,385, herein incorporated by reference in its entirety) by standard cloning techniques.

[0142] In the gamma-retroviral constructs, there is an oPRE sequence 3′ of the expression cassette(s). The retroviral backbone contains long terminal repeats (LTRs) which are located at the 5′- and 3′-end of the cassette. The 5′-LTR contains an SV40 enhancer, RSV promoter, an SFFVp R and U5 region. The 3′-LTR contains an SFFVp U3 region that has a deletion and thus renders the vector self-inactivating (SIN), an SFFV R and U5 region as well as a PolyA signal.

[0143] In the lentiviral construct, there also is an oPRE sequence 3′ of the expression cassette(s). The lentiviral backbone contains long terminal repeats (LTRs) which are located at the 5′- and 3′-end of the cassette. The 5-LTR contains a CMV promoter and an HIV-1 R and U5 region. The 3′-LTR contains an HIV-1 U3 region that has a deletion and thus renders the vector self-inactivating (SIN), an HIV-1 R and U5 region as well as a PolyA signal.

[0144] 2. Titration of Retroviral Supernatants:

[0145] Viral particles encoding the designated vectors are generated by transient transfection of 293T cells (Soneoka et al., Nucleic Acids Research, 1995). In order to determine viral titers, HT1080 fibrosacroma cells are seeded in 12-well plates on day 1, viral supernatants at different dilutions are added to cells on day 2, and three control wells are used to determine cell numbers per well. Three days after transduction, transduction efficiency is analyzed by an intracellular flow cytometry assay detecting AAT protein. To enhance the detection of the protein, cells are treated with GolgiPlug Protein Transport Inhibitor (BD, 555029) for 16 to 24 hours before the staining to prevent the secretion of cytosolic proteins. Cells are permeabilized using BD Cytofix/Cytoperm Fixation and Permeabilization Solution (BD, 554722) according to manufacturer's instructions, and AAT-expressing cells are stained with a FITC-conjugated Anti-alpha 1 Antitrypsin antibody (abcam, ab19170; 1 μL antibody per 100 μL staining reaction and up to 1×10.sup.6 cells, incubation for 20-30 minutes at 4° C. in the dark). Cells are analyzed on a Beckman Coulter FC500 flow cytometer. Only values <25% is AAT positive cells are included in titer calculations.

[0146] Refer FIG. 2 for results.

[0147] 3. Preparation of Human Mesenchymal Stem Cells (MSCs):

[0148] Human MSCs are isolated from bone marrow by plastic adherence and are cultured in growth medium e.g. FBS containing DMEM as described by Pittinger, M. F. (2008) Mesenchymal stem cells from adult bone marrow, In D. J. Prockop, D. G. Phinney, B. A. Bunnell, Methods in Molecular Biology 449, Mesenchymal stem cells, Totowa: Humana Press.

[0149] 4. Genetic Modification of MSCs:

[0150] The transduction of primary MSCs is performed with modifications as described by Murray et al., 1999 Human Gene Therapy. 10(11): 1743-1752 and Davis et al., 2004 Biophysical Journal Volume 86 1234-1242. In detail: 6-well or 12-well cell culture plates (e.g. Corning) are coated with Poly-L-Lysine (PLL) (e.g. Sigma-Aldrich, P4707-50 mL); the PLL solution (0.01%) is diluted to a final concentration of 0.001% with PBS. 1 to 2 mL of diluted PLL are used for each well. The plate is incubated for at least 2 hours at room temperature. After incubation, the plates are washed once with PBS. (Diluted) viral supernatant is added to each PLL-coated well in a final volume of 0.8 to 2 mL. The loaded plate is centrifuged for 2000×g for 30 min at 4° C. The supernatant is then discarded and 1×10.sup.5 MSCs are seeded in one well of a 6-well plate in a volume of 2 mL or 4×10.sup.4 cells are seeded in a well of a 12-well plate in a volume of 1 mL. The plates are incubated at 37° C. with 5% CO.sub.2 for further use.

[0151] 5. Assessment of Transgenic AAT Expression by Intracellular Flow Cytometry:

[0152] To assess transduction efficiency and AAT expression in primary human MSCs, cells are prepared and transduced as described above with multiplicities of infection (MOIs) of 0.25 to 10. Transduced cells are selected with puromycin (Sigma Aldrich, P9620-10 mL, [10 mg/mL], final concentration: 1-5 μg/mL) for 5 to 8 days. Selected cells are analyzed by intracellular flow cytometry as described above.

[0153] All tested vector constructs are able to transduce primary human MSCs, and transgenic AAT is detected by intracellular flow cytometry in all transduced samples. Differences in transduction efficiency as measured by % is AAT positive cells are most likely due to different starting titers of the viral supernatants used for transduction, such that any of the provided cell population upon further isolation and culturing may provide suitable AAT expression. Different expression levels as analyzed by MFI are the result of distinct promoters utilized as well as varied gene cassette constellations.

[0154] Refer to FIG. 3 for results.

[0155] 6. Assessment of Transgenic AAT Expression by ELISA:

[0156] Human MSCs are transduced with the indicated retroviral constructs expressing AAT and the pac gene. The cells are selected with puromycin as described above and 1×10.sup.5 cells are seeded onto 6- or 12-well plates. Supernatants (1-2 mL) are collected after 48 hours and analyzed by ELISA (alpha 1 Antitrypsin (SERPINA1) Human ELISA Kit, abcam, ab108799) according to manufacturer's instructions. Data generated are normalized to 1×10.sup.5 cells and vector copy number (VCN).

[0157] Transgenic AAT expression in primary human MSCs is confirmed in all samples by ELISA. Variances in the amounts expressed are due to distinct promoters utilized in the vectors as well as varied gene cassette constellations, such that each of the examples provided enables sufficient AAT expression at the protein level to obtain a desired effect.

[0158] Refer to FIG. 4 for results.

[0159] 7. Inhibition of Neutrophil Elastase by AAT Expressed from Transduced MSCs:

[0160] Human MSCs are transduced with the indicated retroviral constructs expressing AAT and the pac gene. The cells are selected with puromycin and selected cells are seeded onto 6- or 12-well plates in DMEM without serum. Supernatants (1-2 mL) are collected after 48 hours and analyzed by a Neutrophil Elastase Inhibitor Screening Kit (abcam, ab118971) according to manufacturer's instructions. Supernatant of transduced MSCs is analyzed in different dilutions ranging from non-diluted (1:1) to 1:16 in DMEM without serum. SPCK in different concentrations and medium containing serum (Bio-M and Bio-1) are included as positive controls, DMEM is used as a negative control.

[0161] Inhibition of neutrophil elastase is a functional assay for detecting AAT activity in in vitro. The constructs provided herein show effective inhibition of neutrophil elastase thereby indicating expression of functional AAT from the modified MSCs of the examples.

[0162] Refer to FIG. 5 for results.

[0163] 8. Assessment of Immunomodulatory Effect of AAT Expressed from MSCs on Monocytes:

[0164] Peripheral blood mononuclear cells (PBMCs) are isolated from human blood using ficoll density gradient centrifugation as described by Ivan J. Fuss, Marjorie E. Kanof, Phillip D. Smith, Heddy Zola, 2009 Curr. Protoc. Immunol. 85: 7.1.1-7.1.8. To assess the immunomodulatory effect of AAT-MSCs in vitro, an assay of LPS-induced human monocyte activation is performed as described in Janciauskiene et al., Biochemical and Biophysical Research Communications, 2004. In brief, monocytes are stimulated with lipopolysaccharide (LPS) and the effect of AAT expressed from MSCs on the secretion of proinflammatory cytokines such as TNFalpha and IL-1beta as well as on the expression of anti-inflammatory cytokines such as IL-10 from human monocytes is assessed in supernatants by ELISA.

[0165] When human primary monocytes are cultured in the presence of AAT secreted from genetically modified MSCs (supernatants from MSCs transduced to express AAT), the expression of proinflammatory cytokines (TNFalpha, IL-1beta) in supernatants harvested from monocyte cultures is markedly decreased, whereas there is an increase in the levels of anti-inflammatory cytokines such as IL-10.

[0166] 9. Assessment of Immunomodulatory Effect of AAT-MSC Administration in Animal Models:

[0167] Cells used in vivo experiments are prepared and genetically modified as described above. Transduced cells are selected and expanded further before either cryopreservation or harvest for administration. The cells are either thawed, washed with and re-suspended in PBS or any other suitable buffer prior to injection, or detached from the culture flasks, washed with and re-suspended in PBS or any other suitable buffer, and then injected.

[0168] 10. Bleomycin (BLM)-Induced Pulmonary Fibrosis:

[0169] To test the immunomodulatory and anti-fibrotic effect of AAT-MSCs in vivo, a mouse model of bleomycin-induced pulmonary fibrosis is performed as described in Tashiro et al., Translational Science 2015.

[0170] In brief, BLM lung fibrosis is induced in C57BL/6 mice. After the administration of anesthesia, BLM sulfate (Sigma-Aldrich) dissolved in 50 μL of sterile saline at 2.5 U per kg of body weight is administered by direct intratracheal instillation via intubation. 24 to 72 hours after BLM administration, each animal receives 200 μL either PBS (control), 1×10.sup.6 non-transduced MSCs, or 1×10.sup.6 AAT-transduced MSCs in 200 μL PBS by tail vain injection or intratracheal administration. Serum AAT-levels are monitored by retro-orbital blood collection every second day and subsequent detection of AAT protein by ELISA.

[0171] Mice are killed at 14-21 days after BLM administration.

[0172] Left lung lobes are harvested from mice for protein and messenger RNA (mRNA) analysis. For morphometry and histology studies, right lung lobes are fixed by immersion in 10% neutral-buffered formalin for 24 hours and then transferred to PBS at 4° C. Samples are paraffin embedded, and sections are obtained for hematoxylin-eosin and Masson trichrome staining. Pulmonary fibrosis is assessed using the semiquantitative Ashcroft method on Masson trichrome-stained slides (Ashcroft et al., Journal of Clinical Pathology 1988).

[0173] Refer FIG. 6 for an overview of the experimental design.

[0174] At the time point of 21-day sacrifice, BLM mice without MSC treatment demonstrate lung fibrosis by Ashcroft score, whereas mice treated with either MSCs or AAT-MSCs showed decreased fibrosis. The decrease was more marked in the group receiving MSCs expressing AAT.

[0175] 11. Cyclophosphamide-Accelerated Type 1 Diabetes:

[0176] To assess the effect of AAT-MSCs on the development of diabetes in vivo, a mouse model of cyclophosphamide-accelerated type 1 diabetes is performed (adapted from Brode et al., The Journal of Immunology 2006).

[0177] NOD mice are obtained, where the incidence of diabetes in female mice is 75% by 40 weeks of age. To accelerate and synchronize diabetes, female 8-week-old NOD mice are treated with a single i.p. injection of cyclophosphamide (CY) (200 mg/kg body weight in 0.9% normal saline). Mice are then randomly divided into treatment and control groups. One to five days after cyclophosphamide treatment, each animal receives 200 μL either PBS (control), 1×10.sup.6 non-transduced MSCs, or 1×10.sup.6 AAT-transduced MSCs in 200 μL PBS by tail vain or intraperitoneal injection. Mice are monitored weekly for hyperglycemia until they become diabetic, as defined by two consecutive (>24 hr apart) nonfasting blood glucose levels >240 mg/dl.

[0178] All control mice receiving CY and PBS developed diabetes within 30 days, whereas the onset of diabetes in mice treated with either MSCs or AAT-MSCs was delayed. Interestingly, in mice treated with AAT-MSCs, the delay in development of diabetes was increased by 2 weeks compared to non-modified MSCs.

[0179] 12. MSU/C16.0-Induced Gouty Arthritis:

[0180] To assess the anti-inflammatory effect of AAT-MSCs on gouty arthritis in vivo, a mouse model of MSU/C16.0-induced gouty arthritis is performed (adapted from Joosten et al., Annals of the Rheumatic Diseases 2015).

[0181] Male C57BI/6 mice are obtained from Jackson Laboratories (Bar Harbor, Me., USA) and are used at 10-12 weeks. One to three days prior to induction of gouty arthritis, each animal receives 200 μL either PBS (control), 1×10.sup.6 non-transduced MSCs, or 1×10.sup.6 AAT-transduced MSCs in 200 μL PBS by intraperitoneal administration. Joint inflammation is induced by intra-articular injection of 300 μg MSU crystals mixed with 200 μM C16.0/bovine serum albumin (BSA) in 10 μL PBS into the right knee joint of naive mice. Four hours after intra-articular injection, macroscopic joint swelling is determined. Synovial tissue is isolated and either cultured for 2 hours in tissue culture medium at 37° C. or transferred directly into 200 μL Triton×100 (0.5% in PBS). In addition, knee joints are removed for histology.

[0182] Treatment with either non-modified or AAT-MSCs suppressed MSCU/C16.0 induced joint inflammation; however, inflammation was more markedly decreased when mice were treated with AAT-MSCs.

[0183] 13. Effect of AAT-MSCs on GvHD Prevention in an MHC Matched, Minor Antigen Disparate Murine Transplant Model:

[0184] For the assessment of a possible anti-GvHD effect of AAT-MSCs, a murine transplant model (MHC matched, minor antigen disparate) is performed (adapted from Marcondes et al., Blood 2011).

[0185] C57/BL6J mice (H-2.sup.b; The Jackson Laboratory), 10-14 weeks old with average body weight of 28 g, receive single-dose total body irradiation with 1000cGy followed by intra-tail vein injection of T-cell depleted bone marrow (5×10.sup.6 cells), and CD8+ splenic lymphocytes (0.2×10.sup.6 cells) from C3H.SW-H2b/SnJ donors (H-2bc; The Jackson Laboratory). Mice are randomly divided into treatment and control groups. Mice in the experimental group are given either 1×10.sup.6 non-transduced MSCs or 1×10.sup.6 AAT-transduced MSCs in 200 μL PBS by intraperitoneal or intra-tail vein injection before irradiation and donor cell infusion. Mice in the control group are injected, also intraperitoneally or intravenously, with 200 μL PBS. GvHD is assessed by a standard scoring system (Cooke et al., Blood 1996): Body weights were obtained and recorded on day 0 and weekly thereafter. A weekly clinical index is generated by summation of 5 criteria scores: percentage of weight change, posture (hunching), activity, fur texture, and skin integrity (maximum score=10). Blood samples are collected sequentially for cytokine assays.

[0186] Treatment with either non-modified MSCs or AAT-MSCs results in attenuation or prevention of GvHD and superior survival compared to control mice. Interestingly, the beneficial effect was more prominent in AAT-MSCs compared to native MSCs.