NON-INTEGRATING DNA VECTORS FOR THE GENETIC MODIFICATION OF CELLS

Abstract

The present invention relates to a polynucleotide comprising at least one promoter and an S/MAR element, wherein said S/MAR element is located downstream of said promoter and wherein the nucleic acid sequence of said S/MAR element (S/MAR sequence) comprises at least 3sequence motifs ATTA (SEQ ID NO:1) per 100 nucleotides over a stretch of at most 200 nucleotides; the present invention further relates to a composition and to a host cell comprising said polynucleotide, and to the polynucleotide for use in medicine and for use in treating genetic disease. The present invention also relates to a kit and to a device comprising said polynucleotide, and to methods and uses related to the polynucleotide.

Claims

1. A polynucleotide comprising at least one promoter and an S/MAR element, wherein said S/MAR element is located downstream of said promoter and wherein the nucleic acid sequence of said S/MAR element (S/MAR sequence) comprises at least 3 sequence motifs ATTA (SEQ ID NO:1) per 100 nucleotides over a stretch of at most 200 nucleotides.

2. The polynucleotide of claim 1, wherein said S/MAR element is flanked by a splice donor and a splice acceptor.

3. The polynucleotide of claim 1, wherein said polynucleotide further comprises a coding sequence encoding a polypeptide, preferably a selectable marker, wherein said coding sequence encoding a polypeptide intervenes said promoter and said S/MAR element.

4. The polynucleotide of claim 1, wherein said polynucleotide further comprises a coding sequence encoding a selectable marker (selectable marker sequence), said selectable marker sequence intervening said promoter and said S/MAR element, wherein said promoter and said selectable marker sequence together constitute a selectable marker gene, and wherein said selectable marker is a selectable marker of a eukaryotic cell.

5. The polynucleotide of claim 4, wherein said selectable marker gene is a puromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, or a zeocin resistance gene, preferably is a puromycin resistance gene.

6. The polynucleotide of claim 2, wherein a transcript is transcribed from said promoter, from which transcript the sequence of the S/MAR element is spliced out.

7. The polynucleotide of claim 1, wherein said polynucleotide further comprises a bacterial origin of replication and/or a bacterial selectable marker gene, wherein said bacterial origin of replication and/or bacterial selectable marker gene is/are insulated from the residual sequences comprised in the polynucleotide by the presence of at least one insulation element.

8. The polynucleotide of claim 1, wherein said polynucleotide is devoid of a simian virus 40 (SV40) origin of replication, a bovine papillomavirus (BPV) origin of replication, and an Epstein-Barr virus (EBV) origin of replication.

9. The polynucleotide of claim 1, wherein said polynucleotide replicates episomally in a host cell, preferably wherein episomal replication is stable episomal replication, preferably in a mammalian cell.

10. A composition comprising a polynucleotide according to claim 1, preferably wherein said composition is a pharmaceutical composition.

11. A host cell comprising the polynucleotide according to claim 1.

12. (canceled)

13. (canceled)

14. (canceled)

15. A method for stably transfecting a host cell, comprising a) contacting said host cell with a polynucleotide according to claim 1, and, b) thereby, stably transfecting a host cell.

16. The method of claim 15, wherein said stably transfecting comprises stable episomal replication of the polynucleotide, preferably episomal replication to such an extent that the polynucleotide still is detectable in a host cell population after on average 50 cell divisions.

17. (canceled)

18. (canceled)

19. A method for treating genetic disease in a subject, comprising a) contacting said subject with a polynucleotide according to claim 1, and, b) thereby, treating genetic disease in said subject.

20. The host cell of claim 11, wherein said host cell is a CD34+ Progenitor Cell; a CD61+ Thrombocyte; a CD19+ B-Lymphocyte; a CD14+ Monocyte; a CD15+ Granulocyte; a CD3+ Cytotoxic T-Lymphocyte; a CD3+ Helper T-Lymphocyte; a CD3+ activated T-Lymphocyte, a Tumor infiltrating Lymphocyte, or a Natural Killer (NK) cell.

Description

FIGURE LEGENDS

[0139] FIG. 1: Efficiency of establishment and analysis of the genetically modified cell population: A) A cell culture plate with Crystal Violet stained colonies having formed after 4 weeks selection with Puromycin; the efficiency of vector establishment was approximately 40%; B) FACS detection of GFP fluorescence in Puromycin selected cells; fluorescence is very homogenous and the number of non-fluorescing cells is extremely low; 1=pEPI, 2=pSMARt.

[0140] FIG. 2: Result of plasmid rescue of pS/MARt vectors from established cell populations. Plasmid rescue and restriction analysis following bacterial transformation of total DNA derived from Hek293T cells established with pS/MARt and pEPI Plasmid DNA.

[0141] FIG. 3: Southern blot of pSMARt vectors maintained in selected cells: oligonucleotides hybridizing to the GFP gene of pS/MART were used as probes to detect BamHI restricted vector DNA in extracts from host cells (pS/MARt1 to 3); the non-transfected vector was used as a control (“pS/MARt(+)”).

[0142] FIG. 4: Vector map of pS/MART; ori: bacterial origin of replication, P2A: sequence encoding the self-cleaving 2A peptide from porcine teschovirus-1, apolipoB MAR: S/MAR sequence from the apolipoprotein B gene.

[0143] FIG. 5: Colony forming assay with different versions of pSMARt in Hek293T and HeLa cells

[0144] FIG. 6: pSMARt is efficiently retained in dividing cells also in the absence of continuous selection

[0145] FIG. 7: The efficiency of establishment of pSMARt is independent of the selection marker (A), but the presence of an insulator element before the promoter increases its establishment efficiency (B); FIG. 7A: 1=pEPI, 2 pSMARt-Ele40-GFP-2A-Puro, 3=pSMARt-Ele40-GFP-2A-G418; B: 1=pSMARt-UCOE, 2=pSMARt-Ele40, 3=pSMARt, 4=pEPI

[0146] FIG. 8: pSMARt vectors are efficiently retained in primary human CD3+ cells for over a month.

[0147] FIG. 9: The introduction of splice junctions flanking the SMAR improves the establishment of the vectors in dividing cells. (1) pEPI, (2) pS/MARt, (3) NP, (4) NP-SPlice (1,2,3,4 all with β-inf MAR) (5) pS/MARt, (6) NPsplice (5,6 with ApoLMAR)

[0148] FIG. 10: pSMARt is maintained in mouse embryonic stem cells (mESC) and the presence of the vector does not influence the cells' behavior.

[0149] FIG. 11: pSMARt vectors are active during embryogenesis and differentiation, and they are resistant to epigenetic silencing

[0150] FIG. 12: pSMARt sustains the expression of the transgene GFP during stem-cell differentiation

[0151] FIG. 13: A comparison of CAR-T cells modified with pSMARt vectors and lentivirus; Real Time tumor killing, interferon production and cytotoxicity analysis.

[0152] FIG. 14: Analysis of tumor killing in vivo

[0153] FIG. 15: Analysis of tumor samples and maintenance in vivo of CAR T cells modified with pSMARt and lentivirus; 1=Isotype control, 2=untreated, 3=treated with mock T cells, 4=T cells transduced with lentivirus encoding for the CAR anti human CEA, 5=T cells transfected with pSMARt encodigin for the CAR anti human CEA

[0154] FIG. 16: Expression data from pS/MARt and Nano vectors containing the splicing sequences. Hek293T cell populations established with different version of pS/MARt were analyzed for transgene expression 35 days post DNA delivery and selection in Puromycin (0.5 ug/ml) by Flow Cytometry (A). The relative expression of the transgene GFP was evaluated and normalized to the expression of the housekeeping gene GAPDH (B). The figure shows that the introduction of splicing sequences improve the transgene expression by improving the RNA amount in the cells. Figure (A): (1) pS/MARt, (2) Nano-S/MARt, (3) Nano-S/MARt-splice; Figure (B): (1) pS/MARt, (2) Nano-S/MARt, (3) Nano-S/MARt-splice.

[0155] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

EXAMPLE 1

Efficiency of Establishment and Analysis of the Genetically Modified Cell Population (FIG. 1)

[0156] The efficacy in generating stably expressing cells was evaluated in a colony forming assay using pS/MARt compared to pEPI (FIG. 4, SEQ ID NO:14). Upon DNA delivery, cells positive for GFP transgene expression were isolated via FACS sorting (FACS Aria II) and 100 cells were plated into a 6 cm cell culture dish. They were then cultured for 4 weeks in presence of 0.5 μg/ml Puromycin. After 4 weeks the cells were fixed with PFA and the colonies stained with Crystal Violet. The number of colonies is considered as the efficiency of vector establishment, i.e. the number of colonies forming per number of FACS sorted cells plated. The generation of stable cells lines is very effective with over 40% of transfected cells becoming established (FIG. 1A)). The number of transgene (GFP) expressing cells was estimated by Flow Cytometry. As shown in FIG. 1 b), pS/MARt generates modified populations in which the expression of the transgene is homogenous without significant numbers of negative cells.

[0157] The efficiency of generating stably expressing cells was evaluated by a Flow Cytometry. Hek293T cells were transfected with either pEPI or pS/MARt. The cells were cultured for 35 days and then subjected to FACS analysis to determine the proportion of cells which continued to express the transgene(GFP) and their medium fluorescent intensity. The upper panels of FIG. 1B) show that pS/MARt generates a population of genetically modified cells in which the expression of the transgene is robust and homogenous. This is in contrast to cells transfected with pEPI which show heterogeneous and low levels of transgene expression. The bar charts show that in that in these cellular pools there is no significant population of negative cells in the pS/MARt whilst the pEPI lines have predominantly silenced or lost the transgene expression. Additionally, in the pS/MARt lines median expression of the transgene is substantially higher. Experimental details: cell transfection with JetPEI, selection with 0.5 ug/ml Puro and 1 mg/ml G418 for pEPI.

EXAMPLE 2

Plasmid Rescue of pS/MARt Vectors From Established Cell Populations (FIG. 2)

[0158] We performed DNA rescue experiments to determine the episomal status and the molecular integrity of the vector in mammalian cells.

[0159] Vector DNA was isolated from cells either established with the plasmid pS/MARt or pEPI. These cells were expanded in the presence of the antibiotic Puromycin (0.5 ug/ml) for 1 week and further expanded for at least 30 days without antibiotic. For the plasmid rescue the gDNA from established cells was extracted with the Blood&Tissue DNAeasy kit (Qiagen) and transformed into DH10B E. coli. Bacteria were grown on LB-Agar plates with Kanamycin (50 ug/ml). 12 colonies were grown in liquid LB medium with Kanamycin (50 ug/ml) over night and plasmid DNA was extracted with the MiniprepKit (Qiagen). For the analysis the DNA mini preparations were digested with the restriction enzyme BamHI (Thermo Fisher) for 10 min at 37° C. and the restriction pattern was addressed on a 1% agarose gel. As control the DNA used for transfecting the cells at the beginning of the establishment procedure was digested with the same enzyme and run as a reference. (A) 12 representative samples isolated from pS/MART were analysed and proved to be molecularly equivalent to the original DNA Vector whilst DNA rescued from cells established with pEPI (B) was shown to be molecularly dissimilar to the original vector with smeared bands clearly showing rearrangements of the original DNA.

EXAMPLE 3

pS/MARt Vectors are Maintained Episomally in Modified Cells (FIG. 3)

[0160] To further demonstrate that the pS/MARt vectors were modifying the mammalian cells as an episome, structure was physically determined by Southern Blot analysis. Hek293T cell populations cultured for at least 30 days after DNA transfection were analyzed. The genomic DNA was extracted with the Blood&Tissue DNAeasy kit (Qiagen) and digested over night at 37° C. with the restriction enzyme BamHI (NEB). The total cellular DNA was then separated on a 1% agarose gel and transferred to a nylon membrane. Oligonucleotides corresponding to the vector's GFP gene were used to generate the radioactive probe used to detected the pS/MARt DNA within cellular DNA. The presence in the samples of a single band that has the same size of the control vector demonstrates the episomal status of pS/MARt in the established mammalian cell populations. The absence of smears and/or alternative bands demonstrates that the vectors did not rearrange nor integrate into the cellular genome.

EXAMPLE 3

Efficiency in Generating Stably Expressing Cells (FIG. 5)

[0161] The efficiency in generating stably expressing cells by a range of pS/MARt DNA Vectors harbouring components of the ApoL-MAR and the beta-IFN-MAR was evaluated in a colony forming assay. Upon DNA delivery, cells positive for the expression of the reporter gene GFP were isolated via FACS sorting (FACS Aria II) and 100 were plated into a 6 cm cell culture dish. The cells were then cultured for 4 weeks in the presence of 0.5 g/ml Puromycin. After 4 weeks the cells were fixed with PFA and the colonies stained with Crystal Violet and quantified. The number of colonies is considered as the vector establishment efficiency. The assay shows that vectors engineered with the ApoL MAR, the Core sequence or Fragment 2 are the most efficient in the generation of genetically modified cells.

EXAMPLE 4

Stability in the Absence of Selection (FIG. 6)

[0162] To evaluate whether the insulator sequence could prevent the silencing or loss of DNA vectors we measured the transgene expression of GFP in cells transfected with either (A) pSMARt-insulator-GFP-2a-Puro beta interferon MAR or (B) pSMARt-insulator-GFP-2a-Puro ApoL MAR in the absence of antibiotic selection. Cells were transfected with DNA vectors and cultured in 1 ug/ml Puromycin. After one week the cells were split and the drug was removed form a representative group of cells from each transfection.

[0163] (1) pSMARt-insulator-GFP-2a-Puro beta interferon MAR—cultured in continuous selection

[0164] (2) pSMARt-insulator GFP-2A-Puro beta interferon MAR—selection removed

[0165] (3) pSMARt-insulator-GFP-2a-Puro ApoL MAR—cultured in continuous selection

[0166] (4) pSMARt-insulator GFP-2A-Puro ApoL MAR—selection removed

EXAMPLE 5

Establishment Efficiency (FIG. 7)

[0167] To evaluate the influence of the selection marker on the establishment efficiency we performed colony forming assays as described in FIG. 5. FIG. 7 (A) shows that the establishment efficiency with the DNA vector pS/MARt is independent of the antibiotic selection marker. The selection marker improves establishment efficiency when it is transcriptionally linked to the S/MAR motif. FIG. 7 (B) illustrates improved efficiency of establishment with the additional incorporation of an insulating element between the bacterial backbone and the eukaryotic promoter.

EXAMPLE 6

Stability in Primary Cells (FIG. 8)

[0168] To evaluate the efficacy of our DNA vector system to transfect primary human CD3+ cells and to measure their expression profiles we transfected three variants of the pS/MARt vector system and pEPI encoding GFP and tested their capability for providing sustained expression of the reporter gene GFP in human T cells. The DNA Vectors were delivered to freshly isolated PBMCs via electro-transfer (Nucleofector Device Y, Lonza) and the cells were cultured in presence of IL-2 (5 μg/ml, Bio legend) for 35 days. Every 7 days the cells were checked for the transgene expression and their growth was stimulated via addition in the media of the antibody anti-CD28(Bioegend) and anti-CD3 (Biolegend). pS/MARt carrying the core version of the ApoLMAR is able to generate a higher number of transgene expressing cells gene when compared to the full length ApoL-MAR sequence, to pS/MARt harbouring the βIFN MAR and to pEPI.

EXAMPLE 7

MAR Splicing (FIG. 9)

[0169] To demonstrate that the efficiency of establishment could be improved by the introduction of splicing sequences we generated a series of DNA Vectors with and without the splice donor and acceptor sites flanking the MAR element. To evaluate the influence of the splicing elements on the establishment efficiency we performed colony forming assays as described in FIG. 5.

[0170] The efficiency of establishment of the DNA vectors are significantly improved upon the improvement and minimalization of the bacterial backbone. With the improvements listed previously namely the selection marker and the insulater sequences and its more modern and improved backbone pS/MARt establishes more efficiently than pEPI. In turn minimalization of the backbone FIG. 9 (3) improves this further. The introduction of splicing sequences to remove the S/MAR element from the mRNA nearly doubles the efficiency of this class of vector. This effect can be reproduced with similar compositions of vectors comprising different S/MAR elements (5-6).

EXAMPLE 8

pS/MARt in Stem Cells (FIG. 10)

[0171] To evaluate whether pS/MARt could effectively modify stem cells without molecular damage we generated stable stem cell lines. The E14 mouse embryonic stem cell (mESC) line was established with the pS/MARt-GFP vector. The expression of the reporter gene GFP was measured 1 month after DNA delivery via fluorescent microscopy (A). mESC modfied with pS/MARt-GFP were stained for the most common pluripotency markers showing that the presence of the episomal vector does not alter their pluripotent features.

EXAMPLE 9

pS/MARt in Embryogenesis (FIGS. 11 and 12)

[0172] pSMARt-GFP labeled mESC were injected into blastocysts of C57BL/6 mice, which results in the formation of chimeras. Hematopoietic organs from these chimeras, such as spleen and bone marrow, were stained with a pan blood surface marker (CD45) and their fluorescence analyzed by flow cytometry. A C57BL/6 mouse and a constitutively expressing UBC::GFP mouse, were used as negative and positive controls respectively. (FIG. 11)

[0173] pS/MARt mediates the persistent expression of a transgene during hematopoietic differentiation. mESC were forced to differentiate into HSC and they were analysed by Flow Cytometry before (day 0) and afer (day 6) the differentiation process. Both, parental and labelled cells were successfully differentiated into HSC and cells tagged with pS/MARt-GFP retained the expression of the reporter gene throughout the procedure. (FIG. 12)

EXAMPLE 10

Comparison with Lentivirus (FIG. 13)

[0174] (A) CD3+ cells sorted from two different healthy donors were modified with S/MARt DNA Vectors expressing the CAR272 which provides targeting against the human CEA epitope and their cytolytic activity was confirmed in MCF-7 cells (a breast cancer cell line) with cytotoxic and Interferon-γ releasing assays.

[0175] (B) Engineered T-cells with the S/MARt Vector system expressing the CAR272 shows an improved killing activity when compared to CD3+ cells modified with lentiviruses carrying the same expression cassette.

EXAMPLE 11

Tumor Killing (FIGS. 14 and 15)

[0176] In vivo analysis of CAR-T cells produced with the S/MARt DNA Vector system, lentivirus and the next-generation NanoS/MARt DNA Vector. (FIG. 14)

[0177] NOD/SCID mice (n=6) were inoculated subcutaneously with 2×10.sup.6 HT29 tumor cells. 3×10.sup.5 CAR+ T-cells generated with the S/MARt DNA Vector, lentivirus and the next-generation were injected into the tail vein of each mouse without prior chemo- or radiotherapy at day 7 post tumour cells injection. The tumour targeting efficacy of the modified cells was compared to mock electroporated CD3+ and recorded as tumour growth (A) and mice survival (B).

[0178] The outgrown tumours (isolated from the tumours described above) were explanted, dissociated and analysed for the presence of the CAR target (FIG. 15). The frequency of CAR+ CD3 within tumor masses was analaysed as well as the presence of CAR expressing CD3 in the spleen.

[0179] The remaing tumours comprise only tumour cells that lack the targeted epitope illustrating that the S/MARt treated T-Cells were effective at killing and clearing tumour cells at at least comparable levels to the lentiviral control. Additionally CAR-T positive cells are still detectable infiltrating within the tumour and populating the spleen indicating that the DNA vector is still actively expressing within the transgenic T-Cells.