Patent Application
20240131066
The Instant Application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Jul. 19, 2023 is named “WIS0068US2” and is 124,266 bytes in size. The Sequence Listing does not go beyond the disclosure in the application as filed.
Senescence is a multifaceted cellular response to endogenous and exogenous stress signals that involves the induction of cell cycle arrest to eliminate unwanted cells. A fundamental feature of cell senescence is the senescence-associated secretory phenotype (SASP), which involves the secretion of tissue specific inflammatory, oxidative, and matrix-degrading factors that can attract immune cells and promote matrix rearrangement to eliminate senescent cell populations. However, in persistently damaged or aged tissues, senescent cell clearance can be compromised due to a lack immune cell recruitment, ultimately resulting in tissue dysfunction. To overcome these challenges researchers have looked to eliminate accumulated senescent cell populations that evade immune cell responses by developing antisenescent therapies also known as “senolytic” treatments. While these therapies yield promising therapeutic potential, new approaches for eliminating senescence cells are critically needed for the further understanding and prevention of tissue dysfunction in senescence associated disease pathologies.
Chimeric Antigen Receptor (CAR) T cell therapies redirect T cell specificity and effector potential functions to attack a desired target in an MHC-1 independent manner, bypassing requirements for peptide presentation. In this way, T cells can be engineered to activate against cell surface antigens for several different pathologies such as cancer, HIV, and fibrosis. Amor and colleagues (Nature, 583(7814), pp. 127-132, 2020) recently demonstrated the ability to reprogram CAR T cell effector function to target senescence associated pathologies by targeting the cell surface antigen urokinase Plasminogen Activator Receptor (uPAR). These T cells were manufactured with γ-retroviruses to target uPAR+ cells to eliminate senescent cell in vivo to reduce inflammation in lung and liver fibrosis. These genomes of these cells were not edited by CRISPR-Cas9, which provides new opportunities to increase the potency, specificity, and persistence of T cell therapies.
What is needed are alternative CAR T cell therapies, incorporating CRISPR-Cas9 genome editing, as potent senolytic agents.
In an aspect, an DNA HDR template for a transgene comprising a chimeric antigen receptor (CAR) gene for inserting the transgene into a T cell expressed gene to generate CAR T cells having the composition:
In another aspect, included are plasmids comprising the HDR template described above.
In another aspect, an ex vivo, virus-free method of site-specifically inserting a transgene containing a chimeric antigen receptor (CAR) gene into a T cell expressed gene to generate CAR T cells comprises
The above-described and other features will be appreciated and understood by those skilled in the art from the following detailed description, drawings, and appended claims.
The present disclosure builds on the production of anti-senescence CAR T cell therapies and adapts this technology with CRISPR/Cas9 and homology directed repair (HDR) to integrate a 4.5 kb second-generation anti-uPAR CAR transgene at the human TRAC locus. We describe uPAR CAR T cell product, e.g., a completely virus-free product, featuring precise genomic integration of our CAR and elimination of senescent cells in vitro. Of particular note, there is an increased presence of senescent cells in neurodegenerative diseases and the CAR T therapies described herein are particularly useful to treat neurodegenerative diseases such as Alzheimer's Disease, Down Syndrome, and Parkinson's Disease.
In an aspect, a DNA HDR template for a transgene comprising a chimeric antigen receptor (CAR) gene for inserting the transgene into a T cell expressed gene to generate CAR T cells having the composition:
As used herein, homology arms (HA) are homology arms are complementary to sequences on both sides of the cleavage site in the T cell expressed gene. The homology arms guide insertion of a synthetic DNA sequence into the T cell expressed gene by endogenous DNA repair of the double-stranded DNA cleavage induced by Cas9 RNP. The homology arms are 50 to 3000 nucleotides in length and are complementary to sequences on either side of the cut site in the T cell expressed gene to facilitate incorporation of the synthetic DNA sequence into the genome of the T cell. Small sequence variations (<100 bases) from complementary sequences could be included to enable barcoding or tracking of various cell types or to increase efficiencies of insertion of the synthetic DNA sequence.
In an aspect, the length of the homology arms influences the efficiency of synthetic DNA sequence integration. In an aspect, the homology arms are 400 to 1000 base pairs, specifically 450 to 750 base pairs long.
In an aspect, the left homology arm includes 383 to 588 bp of the TRAC locus directly upstream of the cutsite, and the right homology arm includes 391 to 499 bp of the TRAC locus directly downstream of the cutsite.
The splice acceptor site (SA) assists in the splicing of the synthetic DNA sequence into the transcript generated from the endogenous T cell expressed gene. The site at the 3′ end of an intron typically contains an SA. Therefore, after homology directed repair, the SA in the integrated sequence before the synthetic CAR gene assists in splicing in the CAR and downstream sequences into the endogenous transcript driven by the T cell expressed gene promoter (e.g., TRAC promoter).
A self-cleaving peptide sequence, e.g., T2A, assists in the separation or cleavage of the translated peptide of the protein product encoded by the synthetic DNA sequence from the protein product of the native T cell expressed gene. Exemplary self-cleaving peptides sequences include viral 2A peptides such as a porcine teschovirus-1 (P2A) peptide, a Thosea asigna virus (T2A) peptide, an equine rhinitis A virus (E2A) peptide, or a foot-and-mouth disease virus (F2A) peptide.
An internal ribosome entry site (IRES) is a site that provides initiation of translation from an internal region of the mRNA. An IRES provides co-expression of two proteins from the same mRNA.
As used herein an inducible control sequence is a regulatory sequence which takes advantage of alternative RNA splicing to provide control of protein expression in response to a small molecule inducer. An exemplary inducible control sequence is Xon which is described in Monteys et al., “Regulated control of gene therapies by drug-induced splicing”, Nature, 596, pp. 291-95 (2021). By using the Xon element upstream of our CAR sequence, transcription and subsequent translation of the uPAR binding fragment can be controlled using an oral dosing of the inducer drug treatment LMI070.
uPAR is the receptor for urokinase-type plasminogen activator (uPA), which promotes the degradation of the extracellular matrix components. uPAR expression is increased in many human cancers. As described in Amor et al., “Senolytic CAR T cells reverse senescence-associated pathologies”, Nature, 583, pp. 127-132 (2020), uPAR is induced on the surface of senescent cells. Amor also described uPAR specific CAR T cells prepared using retroviral vectors for the treatment of senescence-associated diseases. These cells drove expression of uPAR by retroviral promoters and did not modify the TRAC gene, resulting in intact TCR protein on the surface and intact signaling by the receptor.
As used herein, a uPAR binding fragment is a polynucleotide encoding a polypeptide that specifically binds uPAR. WO 2020/0160518, incorporated by reference herein for its description of uPAR binding fragments and polypeptides, describes uPAR antigen binding fragments (e.g., scFv).
In an embodiment, the uPAR binding fragment is an extracellular antigen-binding domain (e.g., human scFv) comprising a heavy chain variable (VH) region and a light chain variable (VL) region, optionally linked with a linker sequence, for example a linker peptide, between the heavy chain variable (VH) region and the light chain variable (VL) region. In certain embodiments, the extracellular antigen-binding domain is a human scFv-Fc fusion protein or full length human IgG with VH and VL regions.
In certain non-limiting embodiments, the uPAR binding fragment of the presently disclosed CAR can comprise a linker connecting the heavy chain variable (VH) region and light chain variable (VL) region of the extracellular antigen-binding domain.
In an aspect, the uPAR binding fragment comprises a VHCDRI sequence, a VHCDR2 sequence, and a VHCDR3 sequence of GFTFSNY (SEQ ID NO: 27), STGGGN (SEQ ID NO: 28), and QGGGYSDSFDY (SEQ ID NO: 29); or GFSLSTSGM (SEQ ID NO: 30), WWDDD (SEQ ID NO: 31), and IGGSSGYMDY (SEQ ID NO: 32) respectively. Additionally or alternatively, in some embodiments, the uPAR binding fragment (e.g., scFv) comprises a VLCDRI sequence, a VLCDR2 sequence, and a VLCDR3 sequence of KASKSISKYLA (SEQ ID NO: 33), SGSTLQS (SEQ ID NO: 34), and QQHNEYPLT (SEQ ID NO: 35); RASESVDSYGNSFMH (SEQ ID NO: 36), RASNLKS (SEQ ID NO: 37), and QQSNEDPWT (SEQ ID NO: 38); or KASENVVTYVS (SEQ ID NO: 39), GASNRYT (SEQ ID NO: 40), and GQGYSYPYT (SEQ ID NO: 41), respectively.
Additionally or alternatively, in some embodiments, the amino acid sequence of the VH of the uPAR binding fragment (e.g., scFv) is:
Additionally or alternatively, in some embodiments, the amino acid sequence of the VL of the uPAR binding fragment (e.g., scFv) is:
Additionally or alternatively, in some embodiments, the uPAR binding fragment (e.g., scFv) comprises an amino acid sequence selected from the group consisting of:
In an aspect, the uPAR binding fragment (e.g., scFv) is encoded by a nucleic acid sequence such as:
Additionally or alternatively, in some embodiments, the uPAR binding fragment (e.g., scFv) is encoded by a nucleic acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 36-38. In some embodiments, the uPAR binding fragment (e.g., scFv) is encoded by a nucleic acid that is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 50-52.
In some embodiments, the chimeric antigen receptor comprises a uPAR binding fragment (e.g., a uPA fragment) comprising the amino acid sequence:
Additionally or alternatively, in some embodiments, the uPAR binding fragment (e.g., uPa fragment) comprises an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 53 or SEQ ID NO: 54. In some embodiments, the uPAR binding fragment (e.g., uPa fragment) comprises an amino acid sequence that is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 53 or SEQ ID NO: 54.
Additionally or alternatively, in some embodiments, the uPAR binding fragment (e.g., a uPAR fragment) is encoded by a nucleic acid sequence:
Additionally or alternatively, in some embodiments, the uPAR binding fragment is encoded by a nucleic acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 55 or 56. In some embodiments, the uPAR binding fragment is encoded by a nucleic acid that is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 55 or 56.
In an aspect, the uPAR binding fragment is an antibody fragment. As used herein, the term “single-chain variable fragment” ˜ or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH:VL heterodimer. The heavy (VH) and light chains (VL) are either joined directly or joined by a peptide-encoded linker (e.g., about 10, 15, 20, 25 amino acids), which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL. The linker is may be rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen binding domain. In certain embodiments, the linker comprises amino acids having the sequence GGGGS GGGGS GGGGS (SEQ ID NO: 57).
A specific uPAR binding fragment includes a heavy chain variable fragment and a light chain variable fragment, optionally connected by a linker (SEQ IDs 40-41).
Typically, the antigen-specific extracellular domain (uPAR binding fragment) is linked to the intracellular domain of the CAR by a transmembrane domain, e.g., derived from a CD4, CD8α, CD28, IgG and/or or CD3zeta transmembrane domain. The transmembrane domain traverses the cell membrane, anchors the CAR to the T cell surface, and connects the extracellular domain to the intracellular signaling domain, thus impacting expression of the CAR on the T cell surface. The uPAR binding fragment is linked to the intracellular domain by a hinge domain such as a CD28 or CD8α hinge domain. The hinge domain provides flexibility to the uPAR binding fragment and improves efficacy. CARs may also further comprise one or more costimulatory domain and/or one or more spacer. A costimulatory domain is derived from the intracellular signaling domains of costimulatory proteins that enhance cytokine production, proliferation, cytotoxicity, and/or persistence in vivo. A spacer or hinge connects (i) the antigen-specific extracellular domain to the transmembrane domain, (ii) the transmembrane domain to a costimulatory domain, (Hi) a costimulatory domain to the intracellular domain, and/or (iv) the transmembrane domain to the intracellular domain. For example, inclusion of a spacer domain (e.g., IgG1, IgG2, IgG4, CD28, CD8) between the antigen-specific extracellular domain and the transmembrane domain may affect flexibility of the antigen-binding domain and thereby CAR function. Transmembrane domains, costimulatory domains, and spacers are known in the art. Exemplary costimulatory domains include OX40, 41BB, ICOS, CD27, CD40, CD40L or a TLR.
The first and second secreted factors are a coding sequence for a neurotrophic factor or cytokine. Secreted factors can include neuroprotective, pro-regenerative secreted factors such as APOE2, sAPPa; pro-memory secreted factors such as IL-4, IL-10; growth factors like BDNF, NGF; factors that attract pro-regenerative immune cells such as IL-1, IL-6, TNF-alpha, IFN-gamma; and the like. Exemplary secreted factors include a pro-regenerative secreted factor, a pro-memory secreted factor, growth factor, or a factor that attracts pro-regenerative immune cells SELECTION MARKERS
In an aspect, the synthetic DNA sequence comprises a coding sequence for a selection marker which can be selectable cell surface receptors such as truncated NGFR (tNGFR) or a fluorescent protein such as mCherry, mKate, GFP, BFP, RFP, CFP, YFP, mCyan, mOrange, tdTomato, mBanana, mPlum, mRaspberry, mStrawberry, and mTangerine.
The polyadenylation (polyA) terminator is a sequence-based element that defines the end of a transcriptional unit within the synthetic DNA sequence and initiate the process of releasing the newly synthesized RNA from the transcription machinery. Exemplary polyA terminators are rabbit beta-globin polyA and a bovine growth hormone polyA.
Exemplary sequences of the present disclosure include the following:
Also included herein is a plasmid comprising the virus-free double-stranded HDR template described herein. Exemplary plasmids are non-viral expression vectors such as pUC57 and pUC57-Mini.
In a gene editing method, guide RNAs direct Cas9 nuclease to create a double stranded DNA break at the target locus. DNA repair involving the DNA template containing the synthetic CAR sequence then allows the integration of the CAR described herein into T cells to provide genome-edited T-cells.
In an aspect, an ex vivo, virus-free method of site-specifically inserting a transgene containing a chimeric antigen receptor (CAR) gene into a T cell expressed gene to generate CAR T cells comprises
As used herein, “introducing” means refers to the translocation of the Cas9 ribonucleoprotein and a DNA template from outside a cell to inside the cell, such as inside the nucleus of the cell. Introducing can include transfection, electroporation, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome mediated translocation, transduction with putative non-integrating viruses (e.g., adeno-associated virus, AAV), viral-like particles (VLPs), and the like.
Unmodified T cells include autologous T cells that are collected from a patient, such as a cancer patient, by peripheral blood draw or leukapheresis. Unmodified T cells can also include T cells from allogeneic healthy donors or induced pluripotent stem cells which can be used to produce universal T cells for administration to a patient. T cells are generally modified ex vivo, that is outside of the patient, and then the modified T cells such as CAR T cells are returned to the patient, such as by intravenous infusion, subcutaneous, intratumoral, intraperitoneal or intravenous or intracerebroventricular infusion or intracerebral injection.
Genome editing of the T cells as described herein uses a CRISPR system, or Cas9 ribonucleoprotein. CRISPR refers to the Clustered Regularly Interspaced Short Palindromic Repeats type II system used by bacteria and archaea for adaptive defense. This system enables bacteria and archaea to detect and silence foreign nucleic acids, e.g., from viruses or plasmids, in a sequence-specific manner. In type II systems, guide RNA interacts with Cas9 and directs the nuclease activity of Cas9 to target DNA sequences complementary to those present in the guide RNA. Guide RNA base pairs with complementary sequences in target DNA. Cas9 nuclease activity then generates a double-stranded break in the target DNA.
CRISPR/Cas9 is a ribonucleoprotein (RNP) complex. CRISPR RNA (crRNA) includes a 20 base protospacer element that is complementary to a genomic DNA sequence as well as additional elements that are complementary to the transactivating RNA (tracrRNA). The tracrRNA hybridizes to the crRNA and binds to the Cas9 protein, to provide an active RNP complex. Thus, in nature, the CRISPR/Cas9 complex contains two RNA species.
Guide RNA, or gRNA, can be in the form of a crRNA/tracrRNA two guide system, or an sgRNA single guide RNA. The guide RNA is capable of directing Cas9-mediated cleavage of target DNA. A guide RNA thus contains the sequences necessary for Cas9 binding and nuclease activity and a target sequence complementary to a target DNA of interest (protospacer sequence).
As used herein, a guide RNA protospacer sequence refers to the nucleotide sequence of a guide RNA that binds to a target genomic DNA sequence and directs Cas9 nuclease activity to a target DNA locus in the genome of the T cell such the TRAC gene, a T cell receptor beta subunit constant gene (TRBC), AAVS1 (i.e., PPP1R12C), TET2, FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, CIITA and B2M genes. In some embodiments, the guide RNA protospacer sequence is complementary to the target DNA sequence. “Complementary” or “complementarity” refers to specific base pairing between nucleotides or nucleic acids. Base pairing between a guide RNA and a target region in exon 1 of the TRAC gene can be via a DNA targeting sequence that is perfectly complementary or substantially complementary to the guide RNA. As described herein, the protospacer sequence of a single guide RNA may be customized, allowing the targeting of Cas9 activity to a target DNA of interest.
Any desired target DNA sequence of interest may be targeted by a guide RNA target sequence. Any length of target sequence that permits CRISPR-Cas9 specific nuclease activity may be used in a guide RNA. In some embodiments, a guide RNA contains a 20 nucleotide protospacer sequence.
In addition to the protospacer sequence, the targeted sequence includes a protospacer adjacent motif (PAM) adjacent to the protospacer region which is a sequence recognized by the CRISPR RNP as a cutting site. Without wishing to be bound to theory, it is thought that the only requirement for a target DNA sequence is the presence of a protospacer-adjacent motif (PAM) adjacent to the sequence complementary to the guide RNA target sequence. Different Cas9 complexes are known to have different PAM motifs. For example, Cas9 from Streptococcus pyogenes has a NGG trinucleotide PAM motif; the PAM motif of N. meningitidis Cas9 is NNNNGATT; the PAM motif of S. thermophilus Cas9 is NNAGAAW; and the PAM motif of T. denticola Cas9 is NAAAAC.
A “Cas9” polypeptide is a polypeptide that functions as a nuclease when complexed to a guide RNA, e.g., an sgRNA or modified sgRNA. That is, Cas9 is an RNA-mediated nuclease. The Cas9 (CRISPR-associated 9, also known as Csnl) family of polypeptides, for example, when bound to a crRNA:tracrRNA guide or single guide RNA, are able to cleave target DNA at a sequence complementary to the sgRNA target sequence and adjacent to a PAM motif as described above. Cas9 polypeptides are characteristic of type II CRISPR-Cas systems. The broad term “Cas9” Cas9 polypeptides include natural sequences as well as engineered Cas9 functioning polypeptides. The term “Cas9 polypeptide” also includes the analogous Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 or CRISPR/Cpf1 which is a DNA-editing technology analogous to the CRISPR/Cas9 system. Cpf1 is an RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism is found in Prevotella and Francisella bacteria. Additional Class I Cas proteins include Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas 10d, Case1, Cse 2, Csy 1, Csy 2, Csy 3, GSU0054, Cas 10, Csm 2, Cmr 5, Cas10, Csx11, Csx10, and Csf 1. Additional Class 2 Cas9 polypeptides include Csn 2, Cas4, C2c1, C2c3 and Cas13a.
Exemplary Cas9 polypeptides include Cas9 polypeptide derived from Streptococcus pyogenes, e.g., a polypeptide having the sequence of the Swiss-Prot accession Q99ZW2 (SEQ ID NO: 58); Cas9 polypeptide derived from Streptococcus thermophilus, e.g., a polypeptide having the sequence of the Swiss-Prot accession G3ECR1 (SEQ ID NO: 59); a Cas9 polypeptide derived from a bacterial species within the genus Streptococcus; a Cas9 polypeptide derived from a bacterial species in the genus Neisseria meningitidis (e.g., GenBank accession number YP_003082577; WP_015815286.1 (SEQ ID NO: 60)); a Cas9 polypeptide derived from a bacterial species within the genus Treponema denticola (e.g., GenBank accession number EMB41078 (SEQ ID NO: 61)); and a polypeptide with Cas9 activity derived from a bacterial or archaeal species. Methods of identifying a Cas9 protein are known in the art. For example, a putative Cas9 protein may be complexed with crRNA and tracrRNA or sgRNA and incubated with DNA bearing a target DNA sequence and a PAM motif.
The term “Cas9” or “Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase. Other embodiments of Cas9, both DNA cleavage domains are inactivated. This is referred to as catalytically-inactive Cas9, dead Cas9, or dCas9.
Functional Cas9 mutants are described, for example, in US20170081650 and US20170152508, incorporated herein by reference for its disclosure of Cas9 mutants.
As used herein, the term editing refers to a change in the sequence of the genome at a targeted genomic location. Editing can include inducing either a double stranded break or a pair of single stranded breaks in the genome, such as in a T cell expressed gene. Editing can also include inserting a synthetic DNA sequence into the genome of the T cell at the site of the break(s).
As used herein, a Cas9 RNP that targets a T cell expressed gene comprises a Cas9 protein and a guide RNA that directs double stranded DNA cleavage of the T cell expressed gene. The guide RNA thus includes a crRNA comprising a single-stranded protospacer sequence and a first complementary strand of a binding region for the Cas9 polypeptide, and a tracrRNA comprising a second complementary strand of the binding region for the Cas9 polypeptide, wherein the crRNA and the tracrRNA hybridize through the first and second complementary strands of the binding region for the Cas9 polypeptide. The single-stranded protospacer region of the guide RNA hybridizes to a sequence in the T cell expressed gene, directing cleavage of the T-cell expressed gene to a specific locus of the T cell expressed gene.
Exemplary T cell expressed genes which can be cleaved by the methods described herein include the AAVS1 (i.e., PPP1R12C), TET2, FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, CIITA, B2M, TRAC and TRBC genes, specifically TRAC. The T cell expressed gene-targeted by Cas9 ribonucleoprotein may result in a reduction or elimination of expression of functional TRAC gene product (e.g., knockout of expression of functional TRAC gene product).
In an aspect, the T cell expressed gene is TRAC and wherein the guide RNA targets the 5′ end of the first exon of TRAC. An exemplary guide RNA useful to target the first encoding exon of TRAC comprises SEQ ID NO: 62; CAGGGTTCTGGATATCTGT or SEQ ID NO: 63; GGGAGTCAAAGTCGGTGAAC
In addition to the Cas9 RNP, the virus-free double-stranded HDR template comprising the synthetic DNA sequence is introduced into the T cells.
The genome-edited T cells are then cultured in in xeno-free medium to provide a cultured population of T cells having the synthetic DNA sequence specifically integrated in the T-cell expressed gene locus. The term “xeno” comes from the Greek “xenos” meaning strange. Xeno-free (or xenogeneic-free) therefore means free from “strange” components, or components from a “strange” species (strange being relative to the native species you're working with). In terms of cell culture, this would mean human cell lines can be cultured using human-derived components (like human serum), and it is considered xeno-free, since there is no difference between species.
As used herein culturing the genome-edited T cells in xeno-free medium can include recovery from integration of the synthetic DNA sequence and/or expansion of the edited T cell population.
In an aspect, the CAR T cells produced by the methods described herein have activity against a neurodegenerative disease, stroke, craniocerebral trauma and/or accident, or an elderly patient in need of treatment for aging, for example. Thus, the methods further comprise administering the cultured population of CAR T cells to a patient in need of treatment for a neurodegenerative disease, stroke, craniocerebral trauma and/or accident, or an elderly patient in need of treatment for aging. Exemplary neurodegenerative diseases include Alzheimer's disease, dementia, Parkinson's disease, Lewy body disease, ataxia, Huntington's disease, amyotrophic lateral sclerosis, Down syndrome, and spinal muscular atrophy.
In an aspect, administering the CAR T cells is by intravenous or intracerebroventricular infusion of intracerebral injection.
The invention is further illustrated by the following non-limiting examples.
Cell lines: Primary Human Dermal Fibroblasts adult (HDFa) were purchased from ATCC and maintained in Dulbecco's Modified Eagle Medium high glucose (Gibco) supplemented with 10% Fetal Bovine Serum (Gibco) and 1% penicillin-streptomycin. For drug-induced senescence experiments, trametinib (S2673) and palbociclib (S1116) were purchased from Selleck Chemicals and dissolved in DMSO to yield 10 mM stock solutions, which were stored at −80° C. Cells were treated with MEK inhibitor (25 nM) and CDK4/6 inhibitor (500 nM). The cells were induced for 48 hours, the growth medium was then changed every two days. Cortical Glutamatergic GFP+ Neurons were purchased from BrainXell. These cells were maintained in 50% Dulbecco's Modified Eagle Medium Nutrient Fixture F-12 (Gibco) and 50% Neurobasal Medium (Gibco) supplemented with 2% B27 Supplement (Thermofischer), 1% N2 Supplement (Thermofischer), 0.5 mM Glutamax™ (Gibco), BDNF 10 ng/mL (Peprotech), 10 ng/mL GDNF (Peprotech), 1 ng/mL TGF-β1 (Peprotech), Geltrex® 15 μg/mL (Thermofischer), Neuron Seeding Supplement Day 1 1× (BrainXell), Supplement K 1× (BrainXell). For drug-induced senescence experiments 300 μM Hydrogen Peroxide (Sigma Aldrich) was added to the neuron cultures for 2 hours to induce oxidative stress. After incubation, media was taken off of the cells and replaced with normal glutamatergic neuron culture. Cell lines were maintained in culture at 37° C. in 5% CO2 and tested negative for mycoplasma.
Isolation of primary T cells from healthy donors: This study was approved by the Institutional Review Board of the University of Wisconsin-Madison (#2018-0103), and informed consent was obtained from all donors. Peripheral blood was drawn from healthy donors into sterile syringes containing heparin and transferred to sterile 50 mL conical tubes. Primary human T cells were isolated using RosetteSep™ Human T Cell Enrichment Cocktail (STEMCELL Technologies). T cells were counted using a Countess™ II FL Automated Cell Counter (Thermo Fisher Scientific) with 0.4% Trypan Blue viability stain (Thermo Fisher Scientific) at a 1:1 dilution. T cells were cultured at a final density of 1 million cells/mL in ImmunoCult™—XF T cell Expansion Medium (STEMCELL) supplemented with 200 U/mL IL-2 (Peprotech) and stimulated with ImmunoCult™ Human CD3/CD28/CD2 T cell Activator (STEMCELL) immediately after isolation, per the manufacturer's instructions.
T cell culture: T cells were cultured in ImmunoCult™—XF T cell Expansion Medium at a density of 1 million cells/mL and stimulated with ImmunoCult™ Human CD3/CD28/CD2 T cell Activator (STEMCELL) for 48 hours prior to electroporation. After 24 hours post-electroporation, VFC T cells were transferred without centrifugation to 1 mL of fresh culture medium with 500 U/mL IL-2. T cells were passaged, counted, and adjusted to 1 million/mL in fresh medium+IL-2 on days 5, 7, 9, 11, and 14 after isolation.
Double-stranded DNA HDR template production: Plasmids were generated by Genscript by inserting CAR constructs into a pUC57 vector. VFC-huPAR.28z-2A-mCherry (also termed VFC-huPAR-mCh) and VFC-mCherry (also termed VFC-mCh) plasmids were transformed in 5-alpha competent E. coli (NEB) and purified using the PureYield™ MiniPrep system (Promega). PCR amplicons were generated from plasmid templates using Q5® Hot Start Polymerase (NEB) and pooled into 600 μl reactions for Solid Phase Reversible Immobilization (SPRI) cleanup (6×) using AMPure XP beads according to the manufacturer's instructions (Beckman Coulter). Each of the 600 μl starting products was eluted into 30 μl of water. Bead incubation and separation times were increased to 5 minutes, and elution time was increased to 15 minutes at 37° C. to improve overall yield. PCR products from round 1 cleanup were pooled and subjected to an ethanol precipitation to increase total concentration. Template concentration and purity was quantified using a IMPLEN NanoPhotemeter® N50. Concentrated template products were diluted in UltraPure H20 at a concentration of 2.5 μg/μl according to Nanodrop™ measurements.
SpCas9 RNP preparation: RNPs were produced by complexing a two-component gRNA to SpCas9. In brief, tracrRNA and crRNA were ordered from IDT, suspended in nuclease-free duplex buffer at 100 μM, and stored in single-use aliquots at −80° C. tracrRNA and crRNA were thawed, and 4.15 μl of each component was mixed 1:1 by volume and annealed by incubation at 37° C. for 30 minutes to form a 50 μM gRNA solution in individual aliquots for each electroporation replicate. Recombinant sNLS-SpCas9-sNLS Cas9 (Aldevron, 10 mg/ml, total 3.33 μl) was added to the complexed gRNA at a 1.2:1 molar ratio and incubated for 15 minutes at 37° C. to form an RNP. Individual aliquots of RNPs were incubated for at least 30 seconds at room temperature with HDR templates for each sample prior to electroporation.
T cell nucleofection: Following guidance from the protocols in the art, RNPs and HDR templates were electroporated 2 days after T cell isolation and stimulation. During crRNA and tracrRNA incubation, T cells were centrifuged for 3 minutes at 200 g and counted using a Countess™ II FL Automated Cell Counter with 0.4% Trypan Blue viability stain (Thermo Fisher). 4.13 million T cells were aliquoted and centrifuged for 10 min at 90 g. During cell spin, 8.33 μl of HDR template (total 16.66 μg) per condition were aliquoted to PCR tubes, followed by RNPs (11.66 μl per well) and were incubated for at least 5 minutes. After cell centrifugation, supernatants were removed by vacuum, and cells were resuspended in 80 μl P3 buffer (Lonza), then transferred to PCR tubes containing RNPs and HDR templates, bringing the total volume per sample to 100 μl. Each sample was transferred directly to a 100 μL Nucleocuvette™ Vessel. T cells were electroporated with a Lonza 4D Nucleofector™ with X Unit using pulse code EH115. Immediately after nucleofection, 100 μl of pre-warmed recovery medium with 500 U/mL IL-2 and 25 μl/mL ImmunoCult™ CD3/CD28/CD2 activator was added to each cuvette. Cuvettes were rested at 37° C. in the cell culture incubator for 15 minutes. After 15 minutes, cells were moved to 200 μl total volume of recovery media and equally distributed to 4 wells round bottom 96 well plate.
Flow cytometry Analysis: T cells were stained and analyzed on day 7 of manufacture for mCherry and TCR expression. Ghost Dye™ Red780 was used as a live dead stain to access cell viability. TCR a/b antibody clone IP26 was used to detect TCR knockout in BD Brilliant Stain Buffer (BD Biosciences). All stained samples were run on an Attune™ NxT Flow cytometer (Thermo Fisher Scientific). T cells were stained and analyzed on day 7 of manufacture for mCherry and TCR expression, and day 10 of manufacture for the full Aurora immunophenotyping panel, using fresh cells. Downstream analyses of all spectral cytometry data were performed in FCS Express 7 Software.
In-out PCR: Following guidance from the art, genomic DNA was extracted from 100,000 cells per condition using DNA QuickExtract™ (Lucigen), and incubated at 65° C. for 15 min, 68° C. for 15 min, and 98° C. for 10 min. Genomic integration of the CAR was confirmed by in-out PCR using a forward primer upstream of the TRAC left homology arm, and a reverse primer binding within the CAR sequence. (ATCTTGTGCGCATGTGAGGGGC (SEQ ID NO: 64) and GCAAGCCAGGACTCCACCAACC (SEQ ID NO: 65). PCR was performed according to the manufacturer's instructions using Q5™ Hot Start Polymerase (NEB) using the following program: 98° C. (30 s), 35 cycles of 98° C. (10 s), 67° C. (20 s), 72° C. (2 min), and a final extension at 72° C. (2 min).
In Vitro Cytotoxicity Assays: For
SA-β-Gal Staining: SA-β-gal staining was performed using CHEMICON® Cellular Senescence Assay Kit (cat. KAA002 Millipore Sigma) at a pH 6.0 for human cells. Adherent cells plated in a 12 well plate and fixed with 500 μl Fixing solution (Millipore Sigma) and incubated at room temperature for 10 minutes, washed twice with 1×PBS and stained with freshly prepared 1×SA-β-gal Detection Solution (Millipore Sigma) at 37° C., without CO2 and protected from the light and left overnight. The SA-β-gal Detection Solution was removed and the cells were washed with twice with 1×PBS. Blue stained cells were imaged on a Leica light microscope and three high power fields per well were counted and averaged to quantify the percentage of SA-β-gal+ cells per population.
To avoid the use of viral vectors in our manufacturing process we began by cloning a second generation huPAR CAR sequence with an appended mCherry fluorescent protein with homology arms at the desired cut site for the start of the first encoding exon, exon 6, of the TRAC locus (
Primary human T cells from healthy donors were electroporated with the purified HDR templates and SpyCas9 ribonucleoproteins (RNPs) targeting the human TRAC locus. Cells were recovered for 24 hours at a 1 million/mL density in round-bottom 96-well plates and were expanded in Immunocult™ xeno-free human T cell expansion medium. The cell viability and proliferation of VFC-huPAR-mCh was monitored over 9 days throughout the manufacturing process. Cells were then assayed on day 7 post-isolation to confirm the integration of the VFC-huPAR-mCh CAR T cell products as well as a virus-free CRISPR mCherry only control (VFC-mCh), in place of the huPAR-mCherry CAR sequence. We achieved consistently high genome editing with the dsDNA templates across 2 donors and demonstrated up to 70% knock-in efficiency, with an average of 20% uPAR+ and >90% total TCR-cells, as measured by flow cytometry (
To evaluate the efficiency of the uPAR-mCh CAR T cells in eliminating uPAR+ cells we measured the in vitro potency against senescent induced fibroblasts. Human dermal fibroblasts (HDFa) were plated at 30% confluency and allowed to adhere for 24 hrs, after incubation cells were induced with CDK4/6 and MEK inhibitors. The cells were then stained with SA-β-galactosidase to access for the presence of the senescence associated secretory phenotype (SASP). We performed an impedance assay measuring loss of resistance from induced and non-induced fibroblast populations over a 48 hour period. We observed potent killing 5:1 effector:target ratios. These results demonstrate potent target cell killing of uPAR+ senescent cells through multiple stimuli (
To avoid the use of viral vectors in our manufacturing process we began by cloning a second generation muPAR CAR sequence with an appended a tNGFR selectable marker with homology arms at the desired cut site for the start of the first encoding exon, exon 6, of the TRAC locus (
Primary human T cells from healthy donors were electroporated with the purified HDR templates and SpyCas9 ribonucleoproteins (RNPs) targeting the human TRAC locus. Cells were recovered for 24 hours at a 1 million/mL density in round-bottom 96-well plates and were expanded in Immunocult™ xeno-free human T cell expansion medium. Cells were then assayed on day 7 post-isolation to confirm the integration of the VFC-muPAR-NGFR CAR T cell products. Genomic integration of muPAR-NGFR CAR was confirmed via “in-out” PCR amplification assay on genomic DNA extracted from 100,000 cells from both VFC-muPAR-NGFR and untransfected control cells with primers specific to the TRAC locus and CAR transgene (
To evaluate the efficiency of the muPAR-NGFR CAR T cells in eliminating uPAR+ cells we measured the in vitro potency against mouse senescent induced fibroblasts. Mouse dermal fibroblasts from Ail4 transgenic mice were plated at 30% confluency and allowed to adhere for 24 h, after incubation cells were induced with CDK4/6 and MEK inhibitors. The cells were then stained with SA-β-galactosidase to access for the presence of the senescence associated secretory phenotype (SASP). We observed potent killing of senescent cells at 5:1 effector:target ratio (
Exemplary templates include:
The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms first, second etc. as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers. The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.
While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This application claims priority to U.S. Provisional Application 63/327,189 filed on Apr. 4, 2022, which is incorporated herein by reference in its entirety.
This invention was made with government support under GM119644 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63327189 | Apr 2022 | US |
