REGULATABLE CELL LINES AND METHODS OF USE THEREOF

Abstract

The present invention provides regulatable cell lines with an engineered genome encoding an auxotrophic response system or element, and methods of use thereof. Growth and protein production of the cell lines is controlled by exposure of the cell line to an auxotrophic factor to activate the auxotrophic response system or element. Examples of auxotrophic factors include a nutrient, an enzyme, a moiety that alters pH, a moiety that alters temperature, a non-organic molecule, a non-essential amino acid, an altered concentration of a moiety, and a cellular niche environment. These regulatable cell lines may be used in regenerative medicine and enzyme replacement therapy.

Claims

1. A regulatable human cell line comprising an engineered genome, engineered to comprise or encode an auxotrophic response system, wherein the expression or activity of a gene has been knocked out or downregulated inducing auxotrophy in the regulatable human cell line.

2. The regulatable human cell line of claim 1, wherein the auxotrophic response system or element is responsive to the presence of an auxotrophic factor selected from the group consisting of a nutrient, an enzyme, an altered pH, an altered temperature, a non-organic molecule, a non-essential amino acid, an altered concentration of a moiety, and a niche environment.

3. The regulatable human cell line of claim 2, wherein under normal physiological conditions the nutrient or the enzyme is neither toxic nor bioavailable in concentrations sufficient to sustain the regulatable cell line in humans.

4. The regulatable human cell line of claim 2, wherein the nutrient is selected from the group of genes consisting of: uracil, biotin, nicotinic acid, glutamate(l?), lysine, adenine, ergosterol, glutamic acid, methionine, ethanolamine, dTMP, threonine, Cysteine, L-arginine, D-mannose, praline, arginine, valine, leucine, isoleucine, histidine, heme, leucine, oleic acid, 0.1 mM beta-alanine, methionine, lysine, valine, isoleucine, pantothenic acid, guanine, 0.25 mM spermine, riboflavin, D-glucosamine, 0.25 mM putrescine, thiamine, thiamine(l+), serine, 5-formyltetra hydrofolic acid, glutathione, galactose, nicotinic acid, ornithine, glutamine, 10 uM spermidine, tryptophan, and glutamic acid.

5. The regulatable human cell line of claim 2, wherein the nutrient is uracil or biotin.

6. The regulatable human cell line of claim 1, wherein the gene that has been knocked out or downregulated is selected from the group of genes consisting of: IBA57, AACS, AADAT, AASDHPPT, AASS, AC01, AC02, ACAT1, ACCS, ACCSL, ACO2, ACSS3, ADSL, ADSS, ADSSL1, ALAD, ALAS1, ALAS2, ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH2, AMD1, ASL, ASS1, ATF4, ATFS, AZIN1, AZIN2, BCAT1, BCAT2, CAD, CBS, CBSL, CCBL1, CCBL2, CCS, CEBPA, CEBPB, CEBPD, CEBPE, CEBPG, CH25H, COO6, CPS1, CTH, CYP51A1, DECR1, DHFR, DHFRL1, DHODH, DHRS7, DHRS7B, DPYD, DUT, ETFDH, FAXDC2, FDFT1, FDPS, FDXR, FH, FPGS, G6PD, GCAT, GCH1, GCLC, GFPT1, GFPT2, GLRXS, GLUL, GMPS, GPT, GPT2, GSX2, H6PD, HAAO, HLCS, HMBS, HMGCL, HMGCLL1, HMGCS1, HMGCS2, HOXA1, HOXA10, HOXA11, HOXA13, HOXA2, HOXA3, HOXA4, HOXA6, HOXA7, HOXA9, HOXAS, HOXB1, HOXB10, HOXB11, HOXB12, HOXB13, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB8, HOXB9, HOXC4, HOXC5, HOXC6, HOXC7, HOXC8, HOXC9, HOXD1, HOXD10, HOXD11, HOXD12, HOXD13, HOXD3, HOXD4, HOXD8, HOXD9, HRSP12, HSD11B1, HSD11B1L, HSD17B12, HSD17B3, HSD17B7, HSD17B7B2, HSDL1, HSDL2, ICA1, ICA1P1, ID01, ID02, IL411, ILVBL, IP6K1, IP6K2, IP6K3, IPMK, IREB2, ISCA1, ISCA2, KATNA1, KATNAL1, KATNAL2, KDM1B, KDSR, KMO, KYNU, LGSN, LSS, MARS, MARS2, MAX, MITF, MLX, MMS19, MPC1, MPC1L, MPI, MSM01, MTHFD1, MTHFD1L, MTHFD2, MTHFD2L, MTHFR, MTRR, MVK, MYB, MYBL1, MYBL2, NAGS, ODC1, OTC, PAICS, PAOX, PAPSS1, PAPSS2, PDHB, PDX1, PFAS, PIN1, PLCB1, PLCB2, PLCB3, PLCB4, PLCD1, PLCD3, PLCD4, PLCE1, PLCG1, PLCG2, PLCH1, PLCH2, PLCL1, PLCL2, PLCZ1, PM20D1, PPAT, PSAT1, PSPH, PYCR1, PYCR2, PYCRL, QPRT, RDH8, RPUSD2, SCD, SCDS, SLC25A19, SLC25A26, SLC25A34, SLC25A35, SLC7A10, SLC7A11, SLC7A13, SLC7A6, SLC7A7, SLC7A8, SLC7A9, SLC7AS, SMOX, SMS, SNAPC4, SOD1, SOD3, SOLE, SRM, TAT, TFE3, TFEB, TFEC, THNSL1, THNSL2, TKT, TKTL1, TKTL2, UMPS, UROD, UROS, USF1, USF2, VPS33A, VPS33B, VPS36, VPS4A, and VPS4B.

7. The regulatable human cell line of claim 6, wherein the gene that has been knocked out or downregulated is uridine monophosphate synthetase (UMPS) or holocarboxylase synthetase (HLCS).

8. The regulatable human cell line of claim 1, wherein the regulatable cell line comprises at least one cell type selected from the group consisting of lymphocytes, induced pluripotent stem (iPS) cells, embryonic stem cells, somatic stem cells, haematopoetic stem cells, and peripheral blood mononuclear cells (PBMCs).

9. The regulatable human cell line of claim 1, wherein the regulatable human cell line is a T-cell line.

10. The regulatable human cell line of claim 1, further comprising the engineered genome encoding a therapeutic product that targets at least one selected from the group consisting of a cytokine, an antigen, and a stem cell.

11. The regulatable human cell line of claim 2, wherein the engineered genome comprises a knockout of a gene that encodes a protein that produces or metabolizes the auxotrophic factor.

12. A pharmaceutical composition comprising a regulatable human cell line of claim 1 and a pharmaceutically acceptable excipient.

13-16. (canceled)

17. A method of treating a disease, a disorder, or a condition in a subject, comprising (a) administering to the subject a regulatable human cell line according to claim 1, and (b) administering the auxotrophic factor to the subject in an amount sufficient to promote growth of the regulatable human cell line.

18-21. (canceled)

22. The method of claim 17, wherein the disease, the disorder, or the condition is selected from the group consisting of cancer, Parkinson's disease, graft versus host disease (GvHD), autoimmune conditions, hyperproliferative disorder or condition, malignant transformation, liver conditions, genetic conditions, juvenile onset diabetes mellitus, and ocular compartment conditions.

23. The method of claim 17, wherein the disease, the disorder, or the condition affects at least one system of the body selected from the group consisting of muscular, skeletal, circulatory, nervous, lymphatic, respiratory endocrine, digestive, excretory, and reproductive systems.

24. The method of claim 17, wherein the regulatable human cell line is regenerative.

25. The method of claim 17, further comprising contacting the subject with more than one regulatable human cell line.

26. The method of claim 17, further comprising contacting the subject with more than one auxotrophic factor.

27. The method of claim 17, wherein step (b) comprises localized release of the nutrient or the enzyme.

28. The method of claim 27, wherein the localized release is effected via utilization of a biocompatible device.

29. The method of claim 28, wherein the biocompatible device restricts diffusion of the regulatable human cell line in the subject.

30. The method of claim 17, further comprising removing the auxotrophic factor to deplete therapeutic effects of the regulatable cell line in the subject or to induce cell death in the regulatable human cell line.

31. The method of claim 17, wherein the therapeutic effects comprise at least one selected from the group consisting of: molecule trafficking, inducing cell death, recruitment of additional cells, and cell growth.

32. (canceled)

33. The method of claim 17, wherein the regulatable human cell line is derived from the subject prior to step (a).

34. (canceled)

35. A method of generating a regulatable human cell line according to claim 1, the method comprising the steps of: (a) obtaining a pool of cells, (b) knocking out or downregulating expression of a target gene selected from Table 1 by using a nuclease to introduce an addition, deletion or substitution in a target gene, and (c) screening for auxotrophy.

36. (canceled)

37. The method of claim 35, wherein the target gene is selected from uridine monophosphate synthetase or holocarboxylase synthetase.

38. The method of claim 35, wherein the regulatable human cell line includes at least one type of cell selected from the group consisting of: lymphocytes, induced pluripotent stem (iPS) cells, embryonic stem cells, somatic stem cells, haematopoetic stem cells, and peripheral blood mononuclear cells (PBMCs).

39. The method of claim 35, wherein the nuclease is a CRISPR/Cas nuclease.

40. The method of claim 35, wherein the regulatable human cell line is auxotrophic for a factor listed in Table 1.

41. The method of claim 35, wherein the regulatable human cell line is auxotrophic for uracil or biotin.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0127] FIG. 1 shows that serum is required for optimal recovery post-electroporation. (A) Schematic of assay used to determine optimal electroporation recovery conditions. Following electroporation, cells were supplied with/without serum, 5-fluoroorotic acid (5-FOA), or an exogenous uracil source (uridine). (B) Cell counts by Cytoflex (Beckman Coulter) flow cytometer after 4 days of recovery post electroporation in indicated media conditions. The figure shows cells administered serum, mock edited cells treated with/without 5-FOA with no serum, and uridine monophosphate synthetase (UMPS) knockout cells treated with/without 5-FOA without serum.

[0128] FIG. 2 shows that maintenance and growth of UMPS InDel containing cells requires an exogenous uracil source. (A) Schematic of the procedure used to assay for growth of UMPS or mock edited T cells following electroporation and recovery. (B) TIDE analysis of UMPS InDels in indicated culture conditions. TIDE analysis was performed on sanger sequencing of UMPS locus with oligonucleotides UMPS-O-1 and UMPS-O-2. Performed on day 8. (C) Percentage of alleles analysed by TIDE containing frameshift InDels. Performed on day 8. (D) Predicted absolute numbers of cells at day 8 containing alleles identified by TIDE. (E) Time course of cell counts with/without UMP. (F) Time course of cell counts with/without uridine.

[0129] FIG. 3 shows 5-FOA is less toxic in UMPS targeted cell lines. (A) Schematic of 5-FOA selection procedure. (B-C) Cell counts after 4 days of 5-FOA selection in indicated culture conditions.

[0130] FIG. 4 shows that 5-FOA selected, UMPS targeted cell lines exhibit optimum growth only in the presence of an exogenous uracil source. (A) Schematic of protocol for the demonstration of uracil auxotrophy. Cell cultures were split following 4-day selection in 5-FOA into test media and grown for 4 further days before cell counting. (B-D) Cell counts of 5-FOA selected cells in exogenous uracil (UMP or uridine) containing or deficient media.

EXAMPLES

Example 1: General T Cell Culture Methods

[0131] Buffy coats were obtained and T cells isolated through Ficoll density gradient centrifugation followed by magnetic enrichment using the Pan T cell isolation kit (Miltenyi).

[0132] Cells were cryopreserved in Bambanker medium. After thawing cells were cultured at 37? C., 5% CO.sub.2 in X-Vivo 15 (Lonza) supplemented with or without 5% human serum (Sigma-Aldrich) and 100 IU/ml human recombinant IL-2 (Peprotech) and 10 ng/ml human recombinant IL-7 (BD Biosciences). UMP or Uridine were added at 250 ug/ml. 5-FOA was added at 100 ug/ml to 1 mg/ml. During culture, medium was refreshed every 2 days.

[0133] T cells were activated using immobilized Anti-CD3 (clone OKT3, Tonbo Biosciences) and soluble anti-CD28 (clone CD28.2, Tonbo Biosciences) for 3 days before electroporation. For the experiments in FIGS. 1, 3 and 4, 1.4 million activated T cells were electroporated. For the experiments in FIG. 2, 30,000 activated T cells were electroporated per condition. These cells were resuspended in electroporation solution, mixed with the pre-complexed RNP and electroporated using a 4D-Nucleofector (Lonza) using program EO-115. The RNP consisted of Cas9 protein (Alt-R? S. pyogenes, IDT) at 300 ug/ml and sgRNA using a sgRNA:Cas9 molar ratio of 2.5.

[0134] Genomic DNA was harvested using QuickExtract (Epicentre). Cells were counted on an automated cell counter using Trypan blue staining or on a Cytoflex (Beckman Coulter) flow cytometer using CountBright beads (ThermoFisher) as a reference. Data was analyzed using Excel (Microsoft) and FlowJo (Tree Star).

[0135] Sanger sequencing of the UMPS locus was performed using UMPS-O-1 and UMPS-O-2, with the region amplified using Phusion Hotstart Flex Mastermix (NEB). Sanger sequencing traces were analysed by TIDE (Brinkman et al, 2014) to identify insertions and deletions (InDels) after editing.

[0136] gRNA Sequences (Including PAMs)

TABLE-US-00002 UMPS-7 (SEQIDNO:1) GCCCCGCAGAUCGAUGUAGAGUUUUAGAGCUAGAAA UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGCUUUU

[0137] Sequencing Oligos for UMPS Locus TIDE Analysis:

TABLE-US-00003 UMPS-O-1: (SEQIDNO:2) CCCGGGGAAACCCACGGGTGC UMPS-O-2: (SEQIDNO:3) AGGGTCGGTCTGCCTGCTTGGCT

Example 2: UMPS Editing by Cas9-sgRNA Electroporation in Human T Cells

[0138] T Cells were thawed and cultured, followed by activation and subsequent electroporation with Cas9-UMPS-7 sgRNA RNP as described above. Following electroporation, cells were allowed to recover in medium with or without serum, 5-FOA or an exogenous uracil source (FIG. 1A). Cell survival following electroporation was markedly increased when serum was included in the media (FIG. 1B).

Example 3: Optimal Growth of a Mixed UMPS Edited Population and Maintenance of UMPS Mutations Requires an Exogenous Uracil Source

[0139] T Cells were electroporated and edited as in Example 2, and allowed to recover for a 2 day period in medium+serum+uridine+UMP. On day 0, cells were shifted to UMP, Uridine or ?uracil source media. This experiment does not feature a selection step and thus the resulting population of cells is a heterogeneous mix of wildtype (WT), heterozygous mutant and homozygous mutant cells. The growth of homozygous UMPS mutant cells should be dependent on an exogenous uracil sourceas these should be auxotrophic (FIG. 2A). When UMPS is targeted, InDels are generated in ?55-75% of cells (as assayed by TIDE (Brinkman et al, 2014, Nucleic Acids Res. 42(22):e168)) (FIG. 2B). When the media is uracil deficient, the frameshift generating InDel frequency in the population is reduced (FIG. 2C). This is consistent with the model that any homozygous auxotrophic UMPS mutant cells would be outcompeted in the population by non-auxotrophic heterozygous mutants and WT cells still present after editingresulting in a reduced predicted number of frameshift InDel containing cells (FIG. 2D). The optimal growth of the heterogeneous UMPS edited population is also dependent on the presence of an exogenous source of uracil (FIG. 2E-F). UMP and uridine rescue the growth of UMPS edited cells to the same level as mock edited and CCR5 targeted controls (a control editing locus). This decreased cell growth rate in the absence of exogenous uracil is dependent on UMPS editing, and is not seen in any of the control cells, indicating that edited UMPS causes human T cells to be specifically dependent on uracil supplementation for optimal cell growth.

[0140] It is worth reiterating the UMPS edited population contains unedited or heterozygous cells that are not expected to be auxotrophic, and thus complete lack of growth of UMPS edited cells in uracil deficient media is not expected.

Example 4: 5-FOA Treatment Selects for UMPS Targeted Cells

[0141] 5-FOA selects for uracil auxotrophic cells in other organisms (e.g. Boeke et al. 1984, Mol. Gen. Genet. 197(2):345-6). To investigate the potential utility of 5-FOA for the selection of uracil auxotrophs in human cells, the UMPS gene was targeted in human T cells by Cas9-gRNA complex electroporation followed by recovery (as in Example 2) followed by an assay of resistance to 5-FOA treatment (FIG. 3A). Cells were grown in 5-FOA and a variety of combinations of serum and uracil sources for 4 days before cell counting was performed. Serum, while important for the recovery of cells post electroporation, had no effect on the viability of cells in 5-FOA (FIG. 3B). Uridine and UMP improved the survival of both mock treated and UMPS targeted cells in 5-FOA. This is likely through a competition based mechanism (uridine can reverse 5-fluorouracil toxicity in humans (van Groeningen et al. 1992, Semin. Oncol. 19(2 Suppl 3):148-54)) (FIGS. 3B and C). In all cases, UMPS targeted cells exhibited increased survival vs. mock targeted cells. This data indicates that 5-FOA can be used for the selection of uracil auxotrophic cells in human cell culture.

Example 5: 5-FOA Selected UMPS Targeted Cells Exhibit Uracil Auxotrophy

[0142] To assay whether or not the cells selected for by 5-FOA treatment are uracil auxotrophs, mock or UMPS targeted T cells were exposed to 5-FOA as in Example 4. Following 4 days of 5-FOA selection, the population of cells was split into a uracil containing media (either UMP, uridine or both) and a uracil deficient media. A growth assay was subsequently performed by cell counting after following 4 days incubation in test media (day 8) (FIG. 4A). In all cases, cell growth in the mock targeted cell cultures was negligible and independent of uracil source supplementationindicating successful killing of non-UMPS targeted cells during the 5-FOA selection step (FIG. 4B-D). In the UMPS targeted population, in all conditions cell growth was stimulated by the addition of uracil and poor cell growth was observed in its absence (FIG. 4B-D).

[0143] Taken together, the results of Examples 1-5 indicate that editing of the UMPS locus by Cas9 in human T cells generates cells that are dependent on an exogenous uracil source for optimal cell growth. These results demonstrate that engineered human auxotrophy can be used as a mechanism for controlling the proliferation of T cells or some other cell therapy. In addition, 5-FOA selection of UMPS edited cells provides a useful mechanism for selection of a true auxotrophic population of T cells.

Example 6: Culturing of Stem Cells

[0144] The regulatable cell lines that are the subject matter of the invention herein may include stem cells that were maintained and differentiated using the techniques below as shown in U.S. Pat. No. 8,945,862, which is hereby incorporated by reference in its entirety.

[0145] Undifferentiated hES cells (H9 line from WiCell?, passages 35 to 45) are grown on an inactivated mouse embryonic fibroblast (MEF) feeder layer (Stem Cells, 2007. 25(2): p. 392-401). Briefly, the cell is maintained at an undifferentiated stage on irradiated low-passage MEF feeder layers on 0.1% gelatin-coated plates. The medium is changed daily. The medium consists of Dulbecco's modified Eagle's medium (DMEM)/F-12, 20% knockout serum replacement, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 0.1 mM ?-mercaptoethanol, and 4 ng/ml rhFGF-2 (R&D Systems Inc., Minneapolis). The undifferentiated hES cells are treated by 1 mg/ml collagenase type IV in DMEM/F12 and scraped mechanically on the day of passage. Prior to differentiation, hES cell are seeded onto Matrigel?-coated plates in conditioned medium (CM) prepared from MEF as follows (Nat Biotechnol, 2001. 19(10): p. 971-4). MEF cells are harvested and irradiated with 50 Gy, and are cultured with hES medium without bFGF. CM is collected daily and supplemented with an additional 4 ng/ml of bFGF before feeding hES cells.

Example 7: In Vitro Differentiation of hESC-ECs

[0146] To induce hES cell differentiation, undifferentiated hES cells are cultured in differentiation medium containing Iscove's modified Dulbecco's medium (IMDM) and 15% defined fetal bovine serum (FBS) (Hyclone, Logan, Utah), 0.1 mM nonessential amino acids, 2 mM L-glutamine, 450 ?M monothioglycerol (Sigma, St. Louis, Mo.), 50 U/ml penicillin, and 50 ?g/ml streptomycin, either in ultra-low attachment plates for the formation of suspended embryoid bodies (EBs) as previously described (Proc Natl Acad Sci USA, 2002. 99(7): p. 4391-6 and Stem Cells, 2007. 25(2): p. 392-401). Briefly, hES cells cultured on Matrigel? coated plate with conditioned media are treated by 2 mg/ml dispase (Invitrogen, Carlsbad, Calif.) for 15 minutes at 37? C. to loosen the colonies. The colonies are then scraped off, and transferred into ultra low-attachment plates (Corning Incorporated, Corning, N.Y.) for embryoid body formation.

Example 8: Selection of Auxotrophic Regulatable Cell Lines

[0147] After using the CRISPR/Cas9 platform to generate various gene knockout cell lines. The cells are grown in defined, serum free media, and screened for lack of growth in the media without specific nutrient supplement. Kill curves with different concentrations of supplement vs. control are generated to demonstrate that an exogenously supplied version of the product of the knocked-out gene rescues the auxotrophic phenotype of the cell line. The most promising candidate gene knockouts are engineered in other commonly used cell lines to confirm the effect is universal. CRISPR/Cas9 technology is used to rapidly generate the knockout cell lines. Cell lines with multiple knockouts and mutations may be also generated to provide rapid multiplexed genome engineering and selection (e.g. 5 auxotrophic mutations and 5 antibiotics).

Example 9: In Vivo Analysis

[0148] In vitro validated auxotrophic knockout cell lines may be analyzed in vivo. These cell lines are constrained by toxicity and bioavailability of the auxotrophic factor in humans. The gene knockout cell lines are engineered from human T-cells or any other lymphocyte. Conditional in vitro growth by the cell line is demonstrated in the presence of the auxotrophic factor, and not in the absence of the auxotrophic factor. The cell lines, e.g. human lymphocytes including an auxotrophic response system or element, confirmed to be auxotrophic for the factor may be administered in a mouse model. Only mice consuming the auxotrophic factor supplement are hypothesized to sustain growth of human lymphocytes including the auxotrophic gene deletion that is the auxotrophic response system or element. Further, cell growth stops in vivo upon removal of nutrient from the mouse food source.