WO2021242989A1 - Compositions and methods for inducing cas9 protein expression by use of autonomous pd-1 promoter activity - Google Patents

Abstract

Modified eukaryotic cells that contain a DNA sequence comprising a drug inducible Cre-recombinase expression system, a sequence encoding a Cas9 enzyme, and a conditional promoter that becomes operably linked to the sequence encoding the Cas9 enzyme by function of the Cre-recombinase system, are provided. The disclosure further provides compositions comprising the modified cells and methods of administering the modified cells to an individual in need thereof.

Description

COMPOSITIONS AND METHODS FOR INDUCING CAS9 PROTEIN EXPRESSION BY USE OF AUTONOMOUS PD-1 PROMOTER ACTIVITY

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application no. 63/032,046, filed on May 29, 2020, the disclosure of which is incorporated herein by reference.

FIELD

The present disclosure relates generally to modified cells that can express a CRISPR system, and more particularly to T cells that can express a CRISPR system based on linking expression of the CRISPR enzyme to a PD-1 promoter by inducible recombinase-mediated recombination.

BACKGROUND

Current approaches to adoptive T cell therapy are limited by the difficulty of obtaining sufficient numbers of T cells against targeted antigens with effective in vivo characteristics. There is therefore an ongoing an unmet need for developing compositions and methods to provide improved approaches the generating T cells with desirable characteristics. The present disclosure is pertinent to this need.

SUMMARY

The present disclosure provides modified cells selected from stem cells and leukocytes that are modified to contain a DNA sequence comprising a drug inducible Cre- recombinase expression system, a sequence encoding a Cas9 enzyme, and a conditional promoter that becomes operably linked to the sequence encoding the Cas9 enzyme by function of the Cre-reombinase system. The present disclosure further provides that the modified cells are induced pluripotent stem cells (iPSCs), or wherein the modified cells are leukocytes that are T cells, B cells, antigen presenting cells, or natural killer cells. The disclosure also provides that the conditional promoter is aPdcd-1 promoter. Additionally, the present disclosure provides the modified cells comprise one or more guide RNAs that are functional with the Cas9 enzyme, and which target a DNA sequence of interest in the same cells in which the Cas9 enzyme is expressed. The present disclosure further provides modified cells wherein the drug inducible Cre-recombinase is fused to an estrogen receptor and can be inducible by tamoxifen or a derivative thereof. The present disclosure also provides the modified cells that comprise differentiated progeny of the iPSCs, and wherein the differentiated progeny comprise the Pdcd-1 promoter as the conditional promoter. Further, the present disclosure provides said differentiated progeny in a tumor microenvironment, and wherein the Cre-recombinase system has been induced, and wherein expression of the Cas9 occurs by transcription from the Pdcd-1 promoter.

The current disclosure also provides a method comprising inducing Cre recombinase in the described modified cells such that the promoter becomes operably linked to the DNA sequence encoding the Cas9 gene. The method optionally further comprises introducing one or more suitable guide RNAs into the cells to allow for alteration of a target gene sequence that is targeted by the one or more guide RNAs and wherein the promoter is the Pdcd-1 promoter.

The present disclosure also provides a composition comprising any of the modified cells and a method comprising introducing any of the modified cells into an individual in need thereof.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. A schematic of the Cre-dependent Cas9 Rosa26 targeting vector from Platt et al., (2014) (1).

Figure 2. Plasmid map of the pRetroQ-Cre-ERT2 vector. EF-1 promoter and gag region were individually amplified from Cre-IRES-PuroR (#30205, Addgene) and pRetroQ- Cre-ERT2 (#59701, Addgene), respectively, with the primer sets (Table 1). The two PCR fragments were then cloned into pRetroQ-Cre-ERT2 via Afel-Nhel restriction enzyme sites with the In-Fusion system (Takara).

Figure 3. A flow chart of generation of the pmel-1 iPSCs integrated with both LSL- Cas9-EGFP-Rosa26TV and pRetroQ-Cre-ERT2 vectors.

Figure 4. Fluorescence microscopic (A) and flow cytometric (B) analysis of EGFP expression upon 4-OHT administration (1 mM) in pRetroQ-Cre-ERT2; LSL-Cas9-EGFP- Rosa26TV pmel-1 iPSCs.

Figure 5. Shown in (A) is experimental protocol of adoptive T cell therapy by pmel-1 splenocytes B16F10 tumor bearing C57BL/6). (B) Kinetic analysis of pmel-1 CD8⁺ TILs (top) and splenocytes (bottom) adoptive transferred into C57BL/6 recipients bearing B16F10 tumors. Data show percentage of PD-1, LAG-3, 4-lBB-expressing T-cell subsets based on CX3CR1 -expression (n = 4 mice per group).

Figure 6. Shown in (A) are schematics of the LSL-Cas9-EGFP-PD-1 targeting vectors with (middle row) or without (bottom row) the universal CMV early enhancer/chicken b actin (CAG) promoter. The LSL-Cas9-EGFP-Rosa26 targeting vector (1) is depicted (top) for comparison. Plasmid maps of the LSL-Cas9-EGFP-PD-1 targeting vectors with (B) or without (C) the universal CMV early enhancer/chicken b actin (CAG) promoter.

Figure 7. A flow chart of generation of the pmel-1 iPSCs integrated with either LSL- Cas9-EGFP-PD-1 (with CAG) or LSL-Cas9-EGFP-PD-1 (without CAG) vector.

Figure 8. PCR analysis of individual pmel-1 iPSCs transduced by LSL-Cas9-EGFP- PD-1 (with CAG) and LSL-Cas9-EGFP-PD-1 (without CAG) vectors. Estimated fragment sizes are shown. Asterisks indicate that the undetection of the clone 11- and 12- bands (2849 bp) is possibly from restriction of the PCR condition. The candidate clones based on both left- and right-arm results are shown in the bottom (clone numbers 3, 6, 12).

DETAILED DESCRIPTION

Unless defined otherwise herein, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.

The disclosure includes all polynucleotide and amino acid sequences described herein, and all DNA and RNA sequences that encode any polypeptide as described herein. Each RNA sequence includes its DNA equivalent, and each DNA sequence includes its RNA equivalent. Complementary and anti-parallel polynucleotide sequences are included. Every DNA and RNA sequence encoding polypeptides disclosed herein is encompassed by this disclosure. Amino acids of all protein sequences and all polynucleotide sequences encoding them are also included. Sequences of from 80-99.99% identical to any sequence (amino acids and nucleotide sequences) of this disclosure are included. If reference to an amino acid or nucleotide sequence is made to by way of a database entry, the sequence corresponding to that database entry as it exists on the effective filing date of this application or patent is incorporated herein by reference.

In certain non-limiting embodiments, for which further description is provided below, the present disclosure provides compositions and methods that are intended to overcome imitations of prior methods for generating T cells with desirable characteristics. In certain approaches, the disclosure accordingly provides for using induced pluripotent stem cells (iPSCs) that are intended to provide an ongoing source of autologous T cells. In this regard, iPSC-derived regenerated T cells have potent antitumor efficacy in vitro and in vivo. According to embodiments of this disclosure, iPSCs can be further enhanced by genome engineering and then used to study individual gene function, track cells or endogenous proteins with a knock-in reporter, and correct genetic defects for gene therapy. The present disclosure encompasses all of these features.

In more detail, the present disclosure provides compositions and methods that are used to modify cells such that they express a CRISPR enzyme only when certain conditions are met. In embodiments, expression of a recombinase configures at least one allele of at least one chromosome to express the CRISPR enzyme only under certain cellular contexts, such as within a tumor microenvironment, and/or to be expressed from a promoter that only drives transcription in certain environments. Thus, modified cells that contain controllable expression of a CRISPR enzyme by operation of a recombinase are provided.

In general, the disclosure provides for modifying eukaryotic cells, including but not necessarily limited to T cells, by introducing into the cells using any suitable polynucleotide vector(s) encoding a CRISPR enzyme, and a recombinase. The recombinase is inducible by way of being operably linked to an inducible promoter. Once expression of the recombinase is induced, a sequence encoding the CRISPR enzyme will be positioned in a chromosome such that expression of the CRISPR enzyme is driven by a pre-selected promoter, as described further below. Such a CRISPR enzyme, and suitable guide RNAs, may be considered a “CRISPR system.”

The particular CRISPR enzyme that is expressed subsequent to recombinase-mediated repositioning of the CRISPR enzyme coding sequence is not particularly limited. In embodiments, a Type II CRISPR enzyme is used. In embodiments, the Type II CRISPR enzyme comprises a Cas9 wild type or modified Cas9 enzyme. In embodiments, the CRISPR enzyme creates double stranded chromosome breaks at a particular DNA location, on each strand of a doubles stranded chromosome. In an embodiment, a modified Cas9 enzyme is used so that a single strand DNA cleavage can be used, such as in conjunction with two distinct guide RNAs, each of which will direct the CRISPR single stranded cleavage to opposite strands of the particular chromosomal DNA segment to be modified.

In embodiments, a Cas9 CRISPR enzyme is used. In embodiments, the Cas9 comprises a Cas9 amino acid sequence encoded by Streptococcus pyogenes. In one embodiment, the Cas9 is a variant Cas9 that comprises one or more mutations. In an embodiment, the Cas9 comprises one or more mutations that lessen or eliminate its nuclease activity, but its DNA binding ability is retained. In one embodiment, the mutations comprise a D10A and/or an H840A change in the Streptococcus pyogenes Cas9 amino acid sequence. In an alternative embodiment, Casl2a (formerly Cpfl) may be used.

The CRISPR enzyme may be produced concurrently or sequentially with a detectable protein, such as for detection of cells that express the selected CRISPR enzyme. In embodiments, the CRISPR enzyme and the detectable protein are translated from the same mRNA, and thus may be produced as a fusion protein, or may be produced as separate proteins, such as by the use of an internal ribosome entry site (IRES), or any of a number of self-cleaving signals. Thus, in embodiments, self-cleaving site may be present in the same open reading frame (ORF) as the ORF that encodes the CRISPR enzyme and a detectable protein. A self-cleaving amino acid sequence is typically about 18-22 amino acids long. Any suitable sequence can be used, non-limiting example of which include: T2A (EGRGSLLTCGDVEENPGP) (SEQ ID NO:l); P2A (ATNFSLKQAGDVENPGP) (SEQ ID NO:2); E2A (QCTNYALKLAGDVESNPGP) (SEQ ID NO:3) and F2A (VKQTLNFDLKLAGDVESNPGP) (SEQ ID NO: 4). In embodiments, the detectable protein comprises a fluorescent protein, such as any of green fluorescent protein (GFP), enhanced GFP (EGFP), mCherry, etc., and non-fluorescent proteins such as luciferase and beta- galactosidase.

In embodiments, the recombinase functions under the control of an inducible promoter. The type of recombinase and its recognition sequences are not particularly limited, provided that expression of the recombinase is inducible. In embodiments, the recombinase comprises Cre recombinase, and accordingly may be used with loxP sites. In embodiments, Flp Recombinase is used, and functions in the Flp/FRT system. In embodiments, a Dre recombinase is used, which functions in the Dre-rox system. In embodiments, a Vika recombinase is used, and functions in the Vika/vox system. In embodiments, a Bxbl recombinase is used, and functions with attP and attB sites. In embodiments, a long terminal repeat (LTR) site-specific recombinase (Tre), or other serine recombinases, such as phiC31 integrase, which mediates recombination between two 34 base pair sequences termed attachment sites (att) sites, may be used.

In one embodiment, and as briefly discussed above, the disclosure includes use of a Cre/loxP system, which is a widely used site-specific DNA recombination system derived from bacteriophage PI. Cre recombinase catalyzes a site-specific recombination reaction between two loxP sites and does not require accessory factors. The loxP site is 34bp in length, consisting of two 13bp inverted repeats separated by an 8bp asymmetric spacer sequence. In embodiments, expression of the recombinase is driven from an inducible promoter that is operably linked to the sequence encoding the recombinase.

The DNA sequences of wide variety of inducible promoters for use eukaryotic cells are known in the art, as are the agents that are capable of inducing expression from the promoters. In embodiments, the localization of the recombinase can be regulated. These embodiments include but are not limited to the use of tamoxifen-based relocalization of a recombinase to the nucleus, and/or ligand-induced dimerization of the recombination. Induction of recombinase expression from an inducible promoter, dimerization, and localizing of an existing recombinase to the nuclease are considered to be types of recombinase activation, as used herein.

In embodiments, alternative inducible promoters which drive expression of the recombinase in a controllable manner include the Tet promoter (TRE) which is regulated by tetracycline, anhy dr otetracy cline or doxy cline. In embodiments, the lad-regulated promoter ADHi, which is regulated by IPTG (isopropyl-thio-galactoside) may be used. In embodiments of the disclosure, any inducible promoter that is controlled by tamoxifen or a derivative thereof, such as or 4-hydoxytamoxifen, may be used. In embodiments, a doxorubicin inducible promoter may be used.

In embodiments, the presently provided approach uses constructs, representative embodiments of which are described herein in the text and accompanying figures, wherein induction of expression of a recombinase positions a CRISPR enzyme coding sequence such that it is operably linked with a promoter. An aspect of the disclosure is demonstrated in Example 1 using an EF-1 promoter to express co-express Cas9 with EGFP. Example 2 supports use of the PD-1 promoter, also known as the Pdcd-1 promoter, to selectively drive expression of a CRISPR enzyme, which may also be coupled with expression of a detectable protein. In embodiments, a human or non-human mammalian Pdcd-1 promoter can be used.

In embodiments, a human or mouse promoter may be used. Suitable sequences are known in the art and can be adapted for use in embodiments of this disclosure. In certain implementations, the promoter comprises a segment of one of the sequences from the human PDCD1 (NCBI Reference Sequence: NG 012110.1) or mouse Pdcdl (GenBank: AC167963.5), from which the nucleotide sequences are incorporated herein by reference as they exist in the described database as of the effective filing date of this application or patent.

In embodiments, induction of expression of the recombinase configures the cells to express the Cas enzyme only when transcription from the Pdcd-1 promoter is triggered. In this regard, the Pdcd-1 promoter in its endogenous environment is considered one of the most differentially upregulated gene in the tumor microenvironment compared to periphery after the adoptive transfer of in vitro-activated Pmel-1 splenic CD8+ T cells. In connection with this, accumulating clinical data indicate that tumor-infiltrating CD 8+ T cells in patients have highly elevated expression of PD-1, compared to circulating CD8+ T cells. This strongly supports a TIL-specific PD-1 regulatory mechanism. See, for example, (Blood 114(8): 1537- 44 (2009); J Clin Invest. 124(5):2246-59 (2014); Nat. Med. 22(4):433-8 (2016)), the disclosures of each of which are incorporated herein by reference.

Further, it is known that PD-1 expression supports TCF-1 in Tex precursor cells. In particular, PD-1 has been shown to support the TCF-1+ Tex precursor cells at an early phase of chronic infection (see for example, Immunity 51(5):840-855 (2019)), the disclosure of which is incorporated herein by reference. This supports the presently provided methods, which can be adapted to target and modify Pmel-1 iPS-T cells that would otherwise not persist.

In connection with the Pdcd-1 promoter, at least 10 transcription factor complexes are shown to regulate PD-1 in response to different stimuli, including 8 activators (NFATcl, c- fos/AP-1, Notch, FoxOl, STAT3, STAT4, ISGF3, and NF-kB) and 2 inhibitory molecules (Blimp- 1 and T-bet). This imparts to the present disclosure additional mechanisms to control integrated Cas9 expression by modulating these transcription factor binding to regulatory elements of the Pdcdl locus.

In embodiments, induction of expression of the recombinase results in a recombination event on an integrated construct such that the Pdcdl promoter becomes operably linked to a sequence encoding a CRISPR enzyme, which may be a Cas9 enzyme, as described above. Thus, once the recombinase is induced, a permanent chromosomal rearrangement is achieved such that subsequently, and only when the modified cells are in an environment where transcription driven from the Pdcdl promoter occurs, the Cas9 will be expressed. Accordingly, the disclosure provides for conditional expression of the Cas9, such as when the modified cells are present in a tumor microenvironment, or other environment that promotes expression from the Pdcdl promoter.

In embodiments, function of the Cas9 is coordinated with expression of one or more guide RNAs (gRNAs). The guide RNA(s) may be any suitable form of gRNA, including a single guide RNA (sgRNA), or may require processing before it is functional for DNA cleavage. In embodiments, any gRNA used in the disclosure may be considered a “targeting RNA.” In embodiments, the targeting RNA is selected from a CRISPR RNA (crRNA) and a guide RNA. If the targeting RNA is a crRNA, the system used will further comprises a sequence encoding a separately transcribed trans-activating CRISPR crRNA (tracrRNA) sequence.

In more detail, the sequence of the targeting RNA is not particularly limited, other than by the requirement for it to be directed to (i.e., having a segment that is the same as or complementarity to) a CRISPR site that is specific to a particular location in the same cell where it will function. In this regard, as described briefly above, a target sequence in the modified cells of this disclosure comprises a specific sequence on its 3' end referred to as a protospacer adjacent motif or “PAM”. The PAM is in the targeted DNA, but a targeting RNA directed to a sequence adjacent to the PAM may or may not have the PAM as a component.

In general, the present disclosure is pertinent to target spacer sequences that are subject to cleavage by any Type II CRISPR system, and thus the target sequences conform to the well- known N12-20NGG motif, wherein the NGG is the PAM sequence. It will be recognized that 20 nts is the size of the homology sequence a processed RNA, but, for example, when using a guide RNA that is not processed, the homology sequence can be more than 20 nts, such as up to 40 or more nts. Thus, in embodiments, a targeting RNA used in this disclosure will comprise or consist of a segment that is from 12-40 nucleotides in length. If the encoded targeting RNA, including but not necessarily limited to a pre-crRNA. There are a wide variety of publicly available resources that can be used to design suitable gRNA sequences and such gRNA sequences can be adapted for use with embodiments of the present disclosure.

In embodiments, a gRNA (the targeting RNA) will target any suitable sequence that is intended to be modified. In embodiments, one or more targeting RNAs are provided so that any particular segment of one or both homologous chromosomes can be modified by insertion or deletion (e.g., an indel) or can be knocked out. Alternatively, suitable targeting RNA(s) can be provided to knock-in a DNA segment, such as by including a double stranded repair template to be integrated at a desired location. Gene upregulation is also possible by instead using short gRNAs (14 or 15 base pairs (bp) rather than 20 bp) expressing bacteriophage MS2-binding loops to guide Cas9 to the target locus (PMID: 26436575, 29224783). In embodiments, co-inhibitory receptor genes such as TIGIT (NCBI Gene ID: 201633, 100043314), TIM3 (NCBI Gene ID: 84868, 171285), and LAG3 (NCBI Gene ID: 3902, 16768) are targeted. Additionally, cytokine producing genes are targeted, which may include but it not necessarily limited to IFN-g (NCBI Gene ID: 3458, 15978). Plasmid vectors of these targeting RNAs with drug resistant genes (puromycin (addgene #52963), blasticidin (self manufactured)) can be integrated in iPSCs via a lentiviral method, which enables drug selection. The expression of gRNAs is effective only after Cas9 expression is triggered upon induction of its expression, such as by 4-OHT (tamoxifen or 4-hy doxy tamoxifen) administration. The timing of 4-OHT administration may be after the differentiation of iPSCs into iPS-T cells.

In embodiments, the presently provided approach is used with mammalian cells. In embodiments, the mammalian cells can differentiate into T cells, including but not necessarily limited to CD8+ T cells. In embodiments, the cells are mammalian cells, including but not necessarily limited to non-human mammalian cells, such as mouse cells. In embodiments, the cells are human cells. In embodiments, the cells comprise stem cells. In embodiments, the stem cells are induced stem cells, or are stem cells isolated from an individual. In embodiments, the stem cells are totipotent, pluripotent, or multipotent stem cells. In embodiments, the cells are hematopoietic stem cells. In embodiments, the cells comprise induced pluripotent stem cells (iPSCs). In embodiments, the cells are T cell-derived iPSCs. In embodiments, the iPSCs may be adapted to be antigen-specific T cells. In embodiments, NK cells or their precursors can be modified as described herein.

In embodiments, a construct of this disclosure is inserted into cells using any suitable technique, and expression vector. In embodiments, the expression vector comprises a modified viral polynucleotide, such as from an adenovirus, a herpesvirus, or a retrovirus, such as a lentiviral vector. Polynucleotides can be used directly, or they may be introduced into cells using any of a variety of polynucleotide insertion reagents, such as transfection agents. Non-limiting demonstrations of the disclosure are described below, and use a modified retroviral vector.

In embodiments, insertion of all or a construct described herein is achieved using at least one chromosomal recombination step. In embodiments, the locus of insertion is preselected. In embodiments, the insertion of a construct described herein comprises homologous recombination. In embodiments, the construct is inserted such that a homozygous or heterozygous insertion is produced. Thus, in embodiments, the construct may be inserted at only one preselected allele, which may be confirmed using established techniques. In embodiments, the integration of a construct described herein is at a locus that comprises aPD-1 gene, and/or a Pdcdl promoter locus. In embodiments, to integrate into a preselected locus, the construct may contain homology arms. In embodiments, 5’ and 3’ homology segments that are homologous to segments of a desired locus are included in the construct. In embodiments, the 5’ and 3’ homology segments have a length of from 50-600 bp, inclusive. In embodiments, the homology arms are homologous to a locus that comprises Pdcdl promoter locus. Non-limiting examples of chromosomal modifications are provided below.

In embodiments, introduction of a construct described herein is performed in an individual, or is performed in culture, whereby modified cells that contain the construct in integrated form are introduced into the individual. In embodiments, cells modified according to this disclosure are maintained in culture. In embodiments, the cell culture provides an ongoing supply of modified cells, which can be used for a variety of purposes, including but not limited to research purposes for studying T cell differentiation, activation, interaction with antigen presenting cells, tumors, etc. In embodiments, the T cells are additionally modified to express, for example, a recombinant T cell receptor, a chimeric antigen receptor, or a Bi-specific T-cell engager (BiTE). In embodiments, the T cells or their precursor cells are obtained from an individual, modified, and used to treat one or more conditions for which the individual may be in need of treatment, including but not necessarily limited to any type of cancer, immune disorder, or blood disorder. In embodiments, the T cells are accordingly autologous T cells. In embodiments, the T cells are used in any adoptive immune therapy approach. In embodiments, treatment of an individual with modified T cells as described herein may be combined with any other therapeutic approach, such as checkpoint inhibition, convention chemotherapy, radiation, surgery, etc., and in certain implementation including the cells modified according to this disclosure may produce a synergistic beneficial effect.

The following examples are intended to illustrate but not limit the disclosure.

Example 1

This Example demonstrates that a Cre recombinase-dependent Cas9 expression system (1) can be adopted to obtain inducible Cas9 expression in pmel-1 iPSCs (Figure 1). In this method, the Cas9 expression cassette is knocked into the Rosa26 genomic locus by homologous recombination. The Cas9 expression cassette contains a 3* FLAG-tagged Streptococcus pyogenes Cas9 in frame with a self-cleaving P2A peptide to an enhanced green fluorescent protein (EGFP) to visualize Cas9-expressing cells. In one demonstration, this expression cassette is driven by the universal CMV early enhancer/chicken b actin (CAG) promoter. A loxP-stop-loxP (LSL) cassette is placed behind the CAG promoter to achieve Cas9-2A-EGFP expression only after Cre-mediated recombination (1). For spatiotemporal control of Cre-mediated Cas9-2A-EGFP expression, Cre-driver construct is also required in which Cre recombinase is expressed by a promoter that specifically targets the cell or tissue of interest (2-5). Drug-inducible Cre-driver system is an additional method that enables more precise control of Cre activation in specific cell types (5- 9). CreERT2 consists of Cre protein fused with the estrogen receptor (ER) with a mutated ligand binding domain, which permits synthetic steroids (tamoxifen or 4-hydoxytamoxifen; 4-OHT)-binding dependent activation of Cre recombinase (5,8,9).

One of the ubiquitous promoters, elongation factor-1 (EF-1), has shown to be most effective in promoter-dependent protein expression during mammalian embryonic stem cell (ESC) propagation and differentiation (10-12). We thus devised a Cre-driver vector harboring CreERT2 under the control of EF-1 promoter (Figure 2).

Linearized targeting vector (Cre recombinase-dependent Cas9 expression (1), was electroporated into pmel-1 iPSCs followed by G418 selection for a week (Figure 3). Post- selected single-cell derived iPSC colonies were screened by PCR with primers amplifying both recombinant arms (Table 2). Correctly targeted colonies were subjected to retroviral infection with vectors containing EF-l-CreERT2 followed by puromycin selection. Obtained pmel-1 iPSC clones were split into duplicate wells and cultured in the absence or presence of 4-OHT for 24 hours. EGFP fluorescence were analyzed by phase-contrast fluorescence microscopy (Figure 4A) and flow cytometry (Figure 4B). Although some clones showed basal EGFP expression without 4-OHT treatment (data not shown), the highly 4-OHT- dependent EGFP/Cas9-expressing pmel-1 iPSCs were obtained (Figure 4B). It is expected that these modified pmel-1 iPSCs will be able to differentiate according to known pathways. Thus, this method supports manipulation of Cas9-mediated induction and/or knockout of gene of interest with the use of additional guide RNA expression vectors.

Example 2

This Example demonstrates that the approach described in Example 1 can be modified to provide a method to induce Cas9 protein expression by use of iPST cell-autonomous PD-1 promoter activity.

We previously showed that in vriro-activated iPSC-derived CD8+ T cells have similar phenotype as in vriroactivated pmel-1 splenic CD8⁺ T cells (13), however, those in the tumor microenvironment were not examined. We explored inhibitory receptors (PD-1, LAG-3) and costimulatory receptor (4-1BB) status after adoptive transfer of in vitro- activated pmel-1 splenic CD8⁺ T cells (Figure 5A and 5B). We categorized pmel-1 CD8⁺ T cells based on CX3CR1 -expression levels (CX3CRl^ne§, CX3CRl^int, CX3CRl“), indicative of CD8⁺ T cell- differentiation status (14). Similar to previous study from human melanoma TIL samples (14- 17), pmel-1 CD8⁺ TILs exhibited enhanced expression of those receptors. PD-1 was the most overexpressed compared with spleens, followed by LAG-3 and 4-1BB (Figure 5B). Previous reports showed that intratumoral expression of the PD-1 receptor can guide the identification of the tumor-specific CD8⁺ T cell receptor (TCR) repertoire (14-17). PD-1 expression level seems strictly regulated in both in acute and chronical immune responses by an array of cis-DNA elements, transcription factors, and epigenetic components (18). These findings in part led us to analyze whether utilizing the endogenous PD-1 regulation system may be applicable for pmel-1 iPSC-derived T cells in terms of programmed Cas9 expression. We thus developed targeting vectors harboring homologous recombination arms derived from mouse Pdcd- 1 genomic locus (targeting the first codon of PD-1 gene) with or without CAG promoter upstream of the loxP-stop-loxP (LSL) cassette (Figure 6A-C). These targeting vectors were electroporated into pmel-1 iPSCs (Figure 7). After G418 selection for a week, single-cell derived iPSC colonies were screened by PCR with primers amplifying both recombinant arms (Table 3). By using primer sets either for Pdcd-1 left arm or for Pdcd-1 right arm, we determined that at least some clones likely integrated each construct in the Pdcd-1 locus (Figure 8). This, this example supports the expectation that these modified pmel-1 iPSCs will exhibit Pdcd-1 promoter-dependent Cas9/EGFP expression, particularly in view of the results described in Example 1. It is also expected that this approach will allow manipulation of CRISPR-mediated induction and/or knockout of a gene of interest with the use of additional guide RNA expression vectors in iPSCs and their differentiated progeny.

Table 1: Primer sequences used to amplify the EF-1 promoter region and the gag region.

Table 2: Primer sequences used in genomic PCR screening of LSL-Cas9-Rosa26TV targeting vector.

Table 3: Primer sequences used in genomic PCR screening of LSL-Cas9-Pdcdl targeting vector.

References - this reference listing is not an indication that any of the references are material to patentability.

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12. PLoS One 5(8):el2413 (2010). 13. Cancer Res. 6(12):3473-83 (2016).

14. Immunity 45(6): 1270-1284 (2016).

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Claims

What is claimed is:

1. Modified cells selected from stem cells and leukocytes that are modified to contain a DNA sequence comprising a drug inducible Cre-recombinase expression system, a sequence encoding a Cas9 enzyme, and a conditional promoter that becomes operably linked to the sequence encoding the Cas9 enzyme by function of the Cre-reombinase system.

2. The modified cells of claim 1, wherein the modified cells are induced pluripotent stem cells (iPSCs), or wherein the modified cells are leukocytes that are T cells, B cells, antigen presenting cells, or natural killer cells.

3. The modified cells of claim 1, wherein the conditional promoter is a Pdcd-1 promoter.

4. The modified cells of claim 3, wherein the cells are the iPSCs.

5. The modified cells of claim 4, further comprising one or more guide RNAs that are functional with the Cas9 enzyme, and which target a DNA sequence of interest in the same cells in which the Cas9 enzyme is expressed.

6. The modified cells of claim 5, wherein the drug inducible Cre-recombinase is fused to an estrogen receptor.

7. The modified cells of claim 6, wherein the drug inducible Cre-recombinase is inducible by tamoxifen or a derivative thereof.

8. The modified cells of claim 7, wherein the modified cells comprise differentiated progeny of the iPSCs, and wherein the differentiated progeny comprise the Pdcd-1 promoter as the conditional promoter.

9. The modified cells of claim 8, wherein said differentiated progeny are present in a tumor microenvironment, wherein the Cre-recombinase system has been induced, and wherein expression of the Cas9 occurs by transcription from the Pdcd-1 promoter.

10. A method comprising inducing Cre recombinase in the cells of claim 1 such that the promoter becomes operably linked to the DNA sequence encoding the Cas9 gene, the method optionally further comprising introducing one or more suitable guide RNAs into the cells to allow for alteration of a target gene sequence that is targeted by the one or more guide RNAs.

11. The method of claim 10, wherein the promoter is the Pdcd-1 promoter.

12. The method of claim 11, wherein transcription of the Cas9 from the Pdcd-1 promoter occurs, and wherein the cells are present in a tumor microenvironment.

13. A method comprising inducing Cre recombinase in the cells of any one of claims 2-4 such that the promoter becomes operably linked to the DNA sequence encoding the Cas9 gene, the method optionally further comprising introducing one or more suitable guide RNAs into the cells to allow for alteration of a target gene sequence that is targeted by the one or more guide RNAs.

14. The method of claim 13, wherein the promoter is the Pdcd-1 promoter.

15. The method of claim 14, wherein transcription of the Cas9 from the Pdcd-1 promoter occurs, and wherein the cells are present in a tumor microenvironment.

16. A composition comprising the cells of claim 1.

17. A method comprising introducing cells of claim 1 into an individual in need thereof.