Compositions and methods for restoration of youthful function in CD4 naive t cells

Abstract

Methods and compositions are provided for reducing accelerated T cell differentiation in a CD4 naive T cell (CD4 Tn cell), stimulating (inducing) youthful gene expression in a CD4 Tn cell, and / or reducing cellular age of a CD4 Tn cell. The methods include overexpressing one or more transcription factors (KLF1, KLF4, IKZF1, or any combination thereof) in a CD4 Tn cell. Overexpression can be achieved, e.g., by introducing nucleic acid(s) encoding the transcription factor(s) into a CD4 Tn cell. In some embodiments, the compositions and methods are for use in vivo (i.e., in an individual). In some embodiments, the compositions and methods are for use ex vivo, and overexpressing cells are administered to an individual. In some cases, such an individual has an infectious disease, an autoimmune disease or disorder, or a cancer.

Description

Atty Docket No.: NEWL-003 / 01WQ 42005 / 9COMPOSITIONS AND METHODS FOR RESTORATION OF YOUTHFUL FUNCTION IN CD4 NAIVE T CELLSIncorporation by reference of Sequence Listing provided as an XML File

[0001] A Sequence Listing is provided herewith as a Sequence Listing XML, “NEWL-003-OlWO-Sequence-Listing.xml” created on April 18, 2026, and having a size of 11,438 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.I. Introduction

[0002] There is a need for compositions and methods that enhance CD4 naive T cell function, which can be useful, e.g., in the therapeutic context of infectious disease, autoimmunity, and oncology. Such is provided herein.II. SUMMARY

[0003] Compositions and methods are provided herein for reducing accelerated T cell differentiation of a CD4 naive T cell (CD4 Tn cell), stimulating (inducing) youthful gene expression in a CD4 Tn cell, and / or reducing cellular age of a CD4 Tn cell. In some cases, the compositions and methods are for reducing accelerated T cell differentiation, stimulating (inducing) youthful gene expression, and / or reducing cellular age in a population of CD4 Tn cells, e.g., in vitro (e.g., cells in culture), ex vivo (e.g., recently isolated / harvested from a patient), or in vivo. In some cases, a subject method is a method of treatment. In some cases, a method of treatment results in enhanced CD4 Tn cell function. In some embodiments, the compositions and methods are for use in vivo (i.e., in an individual). In some cases, such an individual has an infectious disease, an autoimmune disease or disorder, or a cancer (e.g., rheumatoid arthritis, a solid tumor cancer, chronic lymphocytic leukemia (CLL), a viral infection, a chronic HIV-1 Infection, a bacterial infection, or any combination thereof).

[0004] The subject methods include (and the subject compositions are for) overexpressing one or more transcription factors in a CD4 naive T cell (CD4 Tn cell). The transcription factors of the disclosure include KLF1, KLF4, IKZF1. The transcription factors can be overexpressed individually, or can be used in combination. As such, in some cases, the one or more transcription factors comprise: KLF1, KLF4, IKZF1, or any combination thereof. In some cases, the one or more transcription factors comprises KLF1. In some cases, the one or more transcription factors comprises KLF4. In some cases, the one or more transcription factors comprises IKZF1. In someAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 cases, the one or more transcription factors comprise IKZF1 and KLF1 (i.e., in some cases, both transcription factors are overexpressed). In some cases, the one or more transcription factors comprise IKZF1 and KLF4 (i.e., in some cases, both transcription factors are overexpressed). In some cases, the one or more transcription factors comprise KLF1 and KLF4 (i.e., in some cases, both transcription factors are overexpressed). In some cases, the one or more transcription factors comprise IKZF1, KLF1, and KLF4 (i.e., in some cases, all three transcription factors are overexpressed).

[0005] In some embodiments, the one or more transcription factors are overexpressed in a CD4 Tn cell by introducing nucleic acid(s) encoding them into the CD4 Tn cell. In some cases, the nucleic acid(s) are mRNAs. In some cases, the nucleic acid(s) are one or more DNAs (e.g., where the nucleotide sequence(s) encoding the transcription factor(s) are operably linked to a promoter such as an inducible promoter). The nucleic acids can be delivered in any convenient way. In some cases, a virus or virus-like particle is used to deliver DNA. In some cases, a lipid nanoparticle (LNP) is used, e.g., LNPs harboring (encapsulating) mRNA(s) or DNA(s) encoding KLF1, KLF4, IKZF1, or any combination thereof.

[0006] A given LNP can include a nucleic acid (e.g., mRNA) encoding one transcription factor, or can include nucleic acids (e.g., mRNAs) encoding more than one of the transcription factors. In some cases, more than one LNP can be used in combination, e.g., in some cases where each LNP harbors a nucleic acid (e.g., mRNA) encoding one of the transcription factors. As an illustrative example, in some cases an LNP can be used that harbors mRNA encoding HCZF1 and also harbors mRNA encoding KLF4 (or KLF1). In other cases, a first LNP harboring mRNA encoding IKZF 1 can be used in combination with a second LNP harboring mRNA encoding KLF4 (or KLF1).

[0007] Compositions for use in the subject methods are provided, as are cells, e.g., CD4 Tn cells, engineered to overexpress the one or more transcription factors. Reagents, compositions, and kits / systems that find use in practicing the subject methods are provided.

[0008] In one embodiment, provided is a CD4 Tn cell engineered to express a T cell receptor of interest (such a cell is referred to herein as a “TCR-T cell”) (autologous or allogeneic), having a subject nucleic acid and / or overexpressing one or more transcription factors.

[0009] Accordingly, disclosed herein are methods (for producing CAR-T cells and / or TCR-T cells (autologous or allogeneic)) in which the CD4 Tn cell in which overexpression occurs is a CAR-T cells or TCR-T cell. For example, in some cases, a subject method includesAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 overexpressing one or more transcription factors in a CD4 Tn cell (e.g., to reduce accelerated T cell differentiation, to induce youthful gene expression where the cell is a CAR-T cell or a TCR-T cell.

[0010] In certain examples, compositions described herein (e.g., one or more nucleic acids encoding the one or more transcription factors, e.g., plasmid(s), viral vector(s), LNPs comprising, and the like)) can be used as manufacturing agents for the production of CAR-T cells and / or TCR-T cells.

[0011] In some cases, the CD4 Tn cell can be a CAR-T cell or a TCR-T cell prior to the overexpressing (meaning, the overexpression of the one or more transcription factors can be in cells that are already TCR-T cells or CAR-T cells prior to the overexpressing). In some cases, the CD4 Tn cells can be converted into a CAR-T cell or a TCR-T cell after said overexpressing (meaning that after initiating overexpression of the one or more transcription factors, the desired receptor (CAR-T or TCR-T) can then be expressed).

[0012] Each of the molecules described (KLF1, KLF4, IKZ l) may be encoded using any of a number of degenerate nucleotide coding sequences that all encode the same functional protein. Additionally, these mRNAs could be formulated as lipid nanoparticles (LNP) for therapeutic expression. LNP-mRNA formulations may be produced using various lipids and different mRNA synthesis procedures such as in vivo transcription, SP6 or T7 based in vitro transcription, or similar. mRNAs might be either unmodified or modified using a different set of alternative nucleotides. Untranslated region sequences (UTRs) for each mRNA might be varied to increase or decrease translational output, increase or decrease mRNA half-life, or to promote cell typespecific translational output. Alternative UTRs might either be sourced from another natural regulatory sequence or a synthetic sequence as designed by a human or computer algorithm.

[0013] Each of the molecules described might also be expressed using an inducible viral gene delivery system, rather than LNP-mRNA. For example, the inventors have demonstrated that a tetracycline inducible lentiviral delivery is sufficient to ameliorate dysfunctional CD4 Tn differentiation using KLFI, KLF4, IKZFl, or combinations of these factors. Similar systems can include, e.g., an adeno-associated virus (AAV) with expression controlled by an inducible splicing event (’’riboswitch”) or protein translation controlled by an endogeneous protease paired with a small molecule protease inhibitor.Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 III. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0015] FIG. 1 : Overview of T cell pooled screening assay. Primary CD3+ T cells were isolated from PBMCs drawn from old (>60 years old; n = 5), young (<30 years old; n = 5) and middle aged donors (40-47 years old; n = 2). The cells were activated using a-CD3 / a-CD28 Dynabeads followed by transduction with a lentiviral library containing between 50 and 150 unique open reading frames (ORFs). four distinct experiments of this type were performed. Each lentiviral pool contained a negative control fluorescent protein (mKate2) and one or more of the TFs of interest: KLF1, KLF4 and IKZF1. The transcription factors were expressed for 4 days followed by a 3 day chase period. Single cell RNA-seq profiles were obtained both at the peak of TF activity (4 days of activation) and after a 3 day chase period. The apparent age of cells harboring each of these TFs was measured using a machine learning model described in Figure 2.

[0016] Fig 2A-2D: Identification of IKZF1 and KLF1 as rejuvenating factors using T cell pooled screens (A) Schematic representation of a novel cell age prediction modeling framework. (B) Schematic representation of an exemplary hold-out donor cross-validation scheme. (C) Receiver operating characteristic curves for the cell age prediction models. The model used for nominating TF sets (NewLimit) is superior to a simple baseline. (D) Cell age effects (lower is values are better) for three TFs. Each point represents a single independent experiment.

[0017] Fig 3A-3B: Aged CD4 naive T cells differentiate more rapidly than young CD4 naive T cells (A) Schematic representation of the CD4 Tn cell differentiation assay. A panel of young and old donors was activated using 10 ng / mL plate bound OKT3 and 2 ug / mL anti-CD28. Following activation, CD4 Tn cells were cultured for an additional 9 days prior to quantification of differentiation state using flow cytometry. (B) The percentage of Tn-like (CD3+CD4+CD27+CD45RA+) and Tcm (CD3+CD4+CD27+CD45RA-) cells for old (n=9 donors) and young (n=10 donors). Each point represents a unique donor. Vertical bars show 95% data intervals. Boxes indicate the interquartile range (25th-75th percentiles) with median (50th percentile) marked by the central line.

[0018] Fig 4A-4B: Transiently expressing TFs that restore youthful gene expression normalizes differentiation rate (A) Schematic description of the CD4 naive T cell differentiation assay withAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 transcription factor (TF) overexpression interventions. Cells are stimulated for one day followed by lenti viral transduction. TFs are then activated by the addition of an inducer molecule for 4 days, followed by a chase period of 5 days where TFs are deactivated. Cells are finally profiled by flow cytometry after staining for naive-like T cell markers (CD27, CD45RA). We found that three TF sets induce more naive-like cells relative to the control (mKate2) as shown in lower panel flow cytometry plots. (B) The fraction of naive-like T cells is quantified for cells treated with each of 17 TFs and 1 negative control (mKate2) following the scheme in (A). KLF4, KLF1, IKZF1 led to significantly higher levels of Tn-like cells than the negative control (q < O.T, linear mixed model formulated as “% Tn ~ (TF treatment) + (1 / Donor)”, FDR was controlled by the Benjamini -Hochberg procedure). Values displayed are the observation minus the random effect learned for each donor.IV. DEFINITIONS

[0019] A DNA sequence that "encodes" a particular RNA is a DNA nucleic acid sequence that is transcribed into RNA. A DNA polynucleotide may encode an RNA (mRNA) that is translated into protein, and the DNA can therefore be said to encode the protein. A DNA may encode an non-coding RNA (ncRNA), i.e., and RNA that is not translated into protein (e.g. tRNA, rRNA, CRISPR / Cas guide RNA).

[0020] A "protein coding sequence" or a sequence that encodes a particular protein or polypeptide, is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' terminus (N-terminus) and a translation stop nonsense codon at the 3' terminus (C-terminus). A transcription termination sequence will usually be located 3' to the coding sequence.

[0021] As used herein, a "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Various promoters, including inducible promoters, may be used to drive the various vectors of the present invention. The promoter may be a constitutively active promoter, i.e. a promoter is active in the absence externally applied agents (e.g., CMV, EFla, beta-Actin), or it may be an inducible promoter (e.g., T7 RNA polymerase promoter, heat shock promoter,Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 Tetracycline-regulated promoter, Steroid-regulated promoter, Metal-regulated promoter, doxycycline-regulated promoter, etc). As used herein, an inducible promoter is a promoter whose activity is regulated by a factor that induces expression, e.g., upon the application of an agent to the cell, (e.g. doxycycline, a tetracycline, and the like), the induced presence of a particular RNA polymerase (e.g., T7 RNA polymerase), and the like.

[0022] The terms "DNA regulatory sequences," "control elements," and "regulatory elements," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and / or regulate transcription of a non-coding sequence (e.g., a guide RNA) or a coding sequence (e g., KLF1, KLF4, IKZF1, a CRISPRa fusion protein, etc.) and / or regulate translation of an encoded polypeptide.

[0023] “Exogenous,” is used herein to refer to something not endogenous to the cell. For example, when an expression vector encoding KLF1, KLF4, IKZF1 and / or a CRISPRa fusion protein is delivered to a cell, the expression vector is exogenous to the cell - the expression vector is an exogenous nucleic acid.

[0024] "Recombinant," as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and / or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms. Alternatively, DNA sequences encoding RNA (e.g., a guide RNA) that is not translated may also be considered recombinant. Thus, e.g., the term "recombinant" polynucleotide or "recombinant" nucleic acid refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., byAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 genetic engineering techniques. Such is usually done to replace a codon with a codon encoding the same amino acid, a conservative amino acid, or a non-conservative amino acid. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.

[0025] A "vector" or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e. an “insert”, may be attached so as to bring about the replication and / or expression of the attached segment in a cell.

[0026] An “expression cassette” comprises a DNA coding sequence operably linked to a promoter. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. The coding sequence can also be said to be operably linked to the promoter.

[0027] The terms “recombinant expression vector,” or “DNA construct” are used interchangeably herein to refer to a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are usually generated for the purpose of expressing and / or propagating the insert(s), or for the construction of other recombinant nucleotide sequences. The insert(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences.

[0028] A cell has been “engineered”, “genetically modified”, "transformed" or "transfected" by exogenous nucleic acid, e.g. a recombinant expression vector or mRNA, when such nucleic acid has been introduced inside the cell. The presence of the exogenous nucleic acid (RNA, DNA) results in permanent or transient change. The exogenous nucleic acid may or may not be integrated (covalently linked) into the genome of the cell (e.g., in some cases when the nucleic acid is DNA). In some cases, the change is transient (e.g., in some cases when mRNA is introduced). In some cases, the change is permanent, e.g., in some cases when an exogenous DNA is integrated into the cell’s genome. A stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA. AAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[0029] Suitable methods of modification (also referred to as “engineering” or “transformation”) include lipid nanoparticle (LNP) delivery, virus or virus-like particle based infection (transduction), transfection, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like. The choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (e.g., in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

[0030] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0031] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0032] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein canAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0034] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0035] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. As such, the articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the polypeptide” includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0036] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. For example, it is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the variousAtty Docket No.: NEWL-003 / 01WO 42005 / 9 embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0037] While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.V. DETAILED DESCRIPTIONCompositions and Methods

[0038] As noted above, provided are compositions and methods for reducing accelerated T cell differentiation of a CD4 naive T cell (CD4 Tn cell), stimulating (inducing) youthful gene expression in a CD4 Tn cell, and / or reducing cellular age of a CD4 Tn cell. Such methods include overexpressing one or more transcription factors.Transcription Factors

[0039] The transcription factors of interest in the present disclosure are KLF1, KLF4, IKZF1:KLF1Synonyms: EKLF; CDAN4A; CDAN4B; EKLF / KLF1< Q13351 MATAETALPSISTLTALGPFPDTQDDFLKWWRSEEAQDMGPGPPDPTEPPLHV KSEDQPGEEEDDERGADATWDLDLLLTNFSGPEPGGAPQTCALAPSEASGAQ YPPPPETLGAYAGGPGLVAGLLGSEDHSGWVRPALRARAPDAFVGPALAPAP APEPKALALQPVYPGPGAGSSGGYFPRTGLSVPAASGAPYGLLSGYPAMYPAP QYQGHFQLFRGLQGPAPGPATSPSFLSCLGPGTVGTGLGGTAEDPGVIAETAPS KRGRRSWARKRQAAHTCAHPGCGKSYTKSSHLKAHLRTHTGEKPYACTWEG CGWRFARSDELTRHYRKHTGQRPFRCQLCPRAFSRSDHLALHMKRHL (SEQ ID NO: 1)mRNA (ORF) used for experiments in the examples section below: SEQ ID NO: 4.Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 KLF4Synonyms: EZF, GKLF< 043474 MRQPPGESDMAVSDALLPSFSTFASGPAGREKTLRQAGAPNNRWREELSHMK RLPPVLPGRPYDLAAATVATDLESGGAGAACGGSNLAPLPRRETEEFNDLLDL DFILSNSLTHPPESVAATVSSSASASSSSSPSSSGPASAPSTCSFTYPIRAGNDPG V APGGTGGGLL YGRE S APPPT APFNL A DI ND VSP SGGF VAELLRPELDP VYIPP QQPQPPGGGLMGKFVLKASLSAPGSEYGSPSVISVSKGSPDGSHPVVVAPYNG GPPRTCPKIKQEAVSSCTHLGAGPPLSNGHRPAAHDFPLGRQLPSRTTPTLGLE EVLSSRDCHPALPLPPGFHPHPGPNYPSFLPDQMQPQVPPLHYQGQSRGFVAR AGEPCVCWPHFGTHGMMLTPPSSPLELMPPGSCMPEEPKPKRGRRSWPRKRT ATHTCDYAGCGKTYTKSSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELT RHYRKHTGHRPFQCQKCDRAFSRSDHLALHMKRHF (SEQ ID NO: 2) mRNA (ORF) used for experiments in the examples section below: SEQ ID NO: 5. IKZF1Synonyms: IK1; LYF1; LyF-1; CVID13; IKAROS; PPP1R92; PRO0758; ZNFN1A1; Hs.54452< Q13422 MDADEGQDMSQVSGKESPPVSDTPDEGDEPMPIPEDLSTTSGGQQSSKSDRVV ASNVKVETQSDEENGRACEMNGEECAEDLRMLDASGEKMNGSHRDQGSSAL SGVGG1RLPNGKLKCD1CG11C1GPNVLMVHKRSHTGERPFQCNQCGASFTQKG NLLRHIKLHSGEKPFKCHLCNYACRRRDALTGHLRTHSVGKPHKCGYCGRSY KQRSSLEEHKERCHNYLESMGLPGTLYPVIKEETNHSEMAEDLCKIGSERSLVL DRLASNVAKRKSSMPQKFLGDKGLSDTPYDSSASYEKENEMMKSHVMDQAI NNAINYLGAESLRPLVQTPPGGSEVVPVISPMYQLHKPLAEGTPRSNHSAQDS A VENLLLL SK AKL VP SEREASP SNSCQD STDTESNNEEQRSGLI YLTNHIAPHA RNGLSLKEEHRAYDLLRAASENSQDALRVVSTSGEQMKVYKCEHCRVLFLDH VMYTIHMGCHGFRDPFECNMCGYHSQDRYEFSSHITRGEHRFHMS (SEQ ID NO: 3)mRNA (ORF) used for experiments in the examples section below: SEQ ID NO: 6.

[0040] The above transcription factors (KLF 1, KLF4, IKZF1) can be overexpressed in a cell (e.g., CD4 Tn cell) individually or in any combination. In some cases, KLF1 is overexpressed. In some cases, KLF4 is overexpressed. In some cases, IKZF1 is overexpressed. In some cases, IKZF1 and KLF1 are overexpressed. In some cases, 1KZF1 and KLF4 are overexpressed. In some cases, KLF1 and KLF4 overexpressed. In some cases, KLF1, KLF4, and IKZF1 are overexpressed.

[0041] Disclosure of the above transcription factors in the context of the present disclosure (e.g., one or more of the transcription factors above, e.g., 1 , 2, or all 3, in compositions (including cells) and methods disclosed herein), is intended to be disclosure of the amino acid sequence as well asAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 nucleic acids that encode such proteins. Cells comprising such polypeptides and nucleic acids are also provided. As will be appreciated, the present disclosure also provides variants of any of the polypeptides and any individual domains therein. In some cases, a variant polypeptide or domain thereof comprises an amino acid sequence having 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% amino acid sequence identity to the parental / reference sequence above, or a functional fragment thereof, where the variant retains the functionality of the parental / reference sequence. As such, the present disclosure includes functional variants of the transcription factors disclosed herein.

[0042] Because of the knowledge of the codons corresponding to the various amino acids, availability of an amino acid sequence of a polypeptide of interest provides a description of all the polynucleotides capable of encoding the polypeptide of interest. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the enzymes disclosed herein. Thus, having identified a particular amino acid sequence, those of ordinary skill in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the polypeptide of interest. In this regard, the present disclosure specifically contemplates each and every possible variation of polynucleotides that could be made by selecting combinations based upon the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide disclosed herein, including the amino acid sequences of SEQ ID NOs.: 1-3.CD4 naive T cells (CD 4 Tn cells)

[0043] The term “naive” refers to immune effector cells that have not encountered antigen. CD4 naive T cells are also referred to herein as “CD4 Tn cells”. As would be understood by one of ordinary skill in the art, all T cells begin as stem cells in the bone marrow. These undifferentiated cells migrate from the bone marrow into the thymus, where they acquire identifying surface markers, such as CD3, CD4, CD8, and TCR (T cells are identified primarily by the presence of the T cell receptor (TCR), CD3, and either CD4 or CD8).

[0044] As would be understood by one of ordinary skill in the art, various strategies can be used to distinguish naive T cells from other T cell types, and any convenient method can be used, e.g., using the presence and absence of specific markers. For example, the table below provides a list of example T cell markers that can be used to distinguish naive T cells. The list includes largelyAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 cell surface proteins, but also intracellular proteins and secreted proteins that can be detected by using exocytosis inhibitors such as brefeldin A or monensin. A combination approach is generally used for identifying naive T cells. In some cases, CD4 Tn cells can be defined as CD4+ / CD27+ / CD45RA+. In some cases, CD4 Tn cells can be defined as CD4+ / CD27+ / CD45RA+ / CD95-. Associated products are offered by a variety of manufacturers and can serve as a useful reference for T cell characterization.

[0045] Table 1.Gene Synonyms Marker Type Protein Type Localization ReferenceCD4 Leu-3 General T Cell Co-Receptor Cell Membrane 1,6,7 CD8 General T Cell Co-Receptor Cell Membrane 1,7 TCRap* T cell General T Cell Receptor Cell Membrane 1receptoralpha-betaIL2RA CD25 Negative Marker Receptor Cell Membrane 2 CD27 TNFRSF7, Naive T Cell, Receptor Cell Membrane 1,3, 4, 5 CD27L MaturationReceptorCD28 Naive T Cell, Co-Receptor Cell Membrane 1,4,5MaturationCD38 Activated T Cell Enzyme Cell Membrane 1,7 CD44 HCAM Negative Marker Cell Adhesion Cell Membrane 3MoleculeCD45RA* Naive T Cell, Receptor Cell Membrane 1,2, 3, 5 Stem MemoryCellCD45RO* Negative Marker Receptor Cell Membrane 1,2,3 SELL CD62L, L- Naive T Cell, Cell Adhesion Cell Membrane 1,2,3selectin Memory T Cell MoleculeAtty Docket No.: NEWL-003 / 01WQ 42005 / 9CD127 IL-7Ra Naive T Cell, Receptor Cell Membrane 1,2MaturationIL6ST CD130, Naive T Cell, Co-Receptor Cell Membrane 1gpl30, MaturationIL6R-betaTNFRSF4 CD 134, T Cell Activation Receptor Cell Membrane 1,40X40CCR7 CD 197 Naive T Cell, Receptor Cell Membrane 1,5,7 Regulatory TCellICOS CD278 Maturation Co-Receptor Cell 1,4Membrane, SecretedFOXP1 Naive T Cell, DNA-Binding Nucleus 1,6Maturation ProteinLEF1 Naive T Cell, DNA-Binding Nucleus 1,6Maturation ProteinTCF7 Naive T Cell, DNA-Binding Nucleus 1,6Maturation ProteinIL2 Activated T Cell Cytokine Secreted 1 HLA-DR* Negative Marker MHC Class II Cell Membrane 1ComponentFAS CD95, Negative Marker Receptor Cell Membrane 1APT1Note: *Some markers are protein isoforms or multi-subunit protein complexes. Information on Protein Type and Localization is from UniProt.org (for human genes only).

[0046] References for the Table aboveAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 1. Mousset CM, Hobo W, Woestenenk R, Preijers F, Dolstra H, van der Waart AB. Comprehensive Phenotyping of T Cells Using Flow Cytometry. Cytometry A.2019;95(6):647-654. doi:10.1002 / cyto.a.237242. Golubovskaya V, Wu L. Different subsets of T cells, memory, effector functions, and CAR-T immunotherapy. Cancers (Basel). 2016;8(3):36.doi : 10.3390 / cancers80300363. Vrisekoop N, den Braber I, de Boer AB, et al. Sparse production but preferential incorporation of recently produced naive T cells in the human peripheral pool. Proc Natl Acad Sci U S A. 2008;105(16):6115-6120. doi:10.1073 / pnas.0709713105 4. Hutten TJA, Norde WJ, Woestenenk R, et al. Increased coexpression of PD-1, TIGIT, and KLRG-1 on tumor-reactive CD8+ T cells during relapse after allogeneic stem cell transplantation. Biology of Blood and Marrow Transplantation.2018;24(4):666-677. doi: 10.1016 / j .bbmt.2017.11.0276. Durek P, Nordstrom K, Gasparoni G, et al. Epigenomic profiling of human CD4+ T cells supports a linear differentiation model and highlights molecular regulators of memory development. Immunity. 2016;45(5): 1148-1161.doi: 10.1016 / j .immuni.2016.10.0227. Kumar BV, Connors T, Farber DL. Human T cell development, localization, and function throughout life. Immunity. 2018;48(2):202-213.doi: 10.1016 / j .immuni.2018.01.007Overexpression

[0047] Subject methods include inducing one or more of the above transcription factors in a CD4 Tn cell, thereby eliciting a desired change in the cell, e.g., reducing accelerated T cell differentiation, stimulated youthful gene expression, and / or reducing cellular age. In some cases, the one or more transcription factors are overexpressed in a population of CD4 Tn cells, e.g., in some cases the CD4 Tn cells are ex vivo (e.g., were recently harvested / isolated from a patient), and in some such cases are present in vivo. As an example, a nucleic acid (e.g., mRNA) encoding the one or more transcription factors can be introduced into a population of CD4 Tn cells (e.g., via contacting the population with one or more LNPs or one or more a viruses).

[0048] The term “overexpression” and “overexpressing” is used herein to refer to the act of increasing expression of a protein (i.e., increasing the amount / level of protein present) in a cell. The increase is relative to the expression / amount / level present prior to the act of overexpression.Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 Thus, the act of stimulating / inducing expression of a protein is considered overexpressing that protein. One of ordinary skill in the art will understand how to achieve overexpression of a desired protein and any convenient method can be used. For example, overexpression can be achieved by introducing a nucleic acid (e.g., a DNA or an mRNA) encoding a protein of interest (e.g., a transcription factor) into a cell. In some cases, overexpression is long-term, e.g., using DNA that can integrate into the genome of the targeted cell. In some cases, overexpression is transient, e g., using mRNA or using a DNA in which the nucleotide sequence of interest is operably linked to an inducible promoter.

[0049] As would be understood to one of ordinary skill in the art, the one or more transcription factors (KLF1, KLF4, IKZF1, or any combination thereof) and / or a CRISPRa fusion protein can be introduced into a cell directly as protein. They can also be introduced as a nucleic encoding the protein (e.g., an RNA or a DNA such as an expression vector). In some cases, the one or more transcription factors and / or a CRISPRa fusion protein is introduced as an mRNA encoding the protein (and the cell translates the mRNA into protein). In some cases, the one or more transcription factors and / or a CRISPRa fusion protein is introduced as a DNA encoding the protein (and the cell expresses the protein via RNA transcription and translation into protein).

[0050] For example, in some cases, overexpression is achieved by introducing one or more nucleic acids encoding the desired protein(s) (KLF1, KLF4, IKZF1, or any combination thereof) into the cell. In some cases, the introduced nucleic acid(s) is mRNA. In some cases, the introduced nucleic acid(s) is DNA that includes a promoter operably linked to the nucleotide sequence(s) encoding the protein(s) (e.g., KLF1, KLF4, IKZF1, or any combination thereof).

[0051] Regardless of whether mRNA is DNA is used, in some cases, a nucleotide sequence encoding the one or more transcription factors (KLF1, KLF4, IKZF1, or any combination thereof) is codon optimized. This type of optimization can entail a mutation of a transcription factorencoding nucleotide sequence to mimic the codon preferences of the intended host organism or cell while encoding the same protein. Thus, the codons can be changed, but the encoded protein remains unchanged. For example, if the intended target cell was a human cell, a human codon-optimized nucleotide sequence can be used. In some cases, even though the nucleotide sequences are human sequences and are intended to be used in human cells (e.g., CD4 Tn cells), the sequences can be codon optimized to modulate (e.g. enhance) translation efficiency. Codon usage tables are readily available, and codon optimization is also commercially available.Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 mRNA

[0052] As noted above, in some cases, overexpression is achieved using mRNA. In some embodiments, an mRNA encoding a transcription factor is not a modified mRNA. In other embodiments, an mRNA encoding a transcription factor is a modified mRNA. Such modifications can be used, e.g., to modulate (i.e., increase or decrease) translational output, increase or decrease mRNA half-life, or to promote cell type-specific translational output.

[0053] For example, in some cases, an mRNA includes pseudouridine, 5-methylcytosine (m’C), N6-methyladenosine (m6A), N^methyladenosine (mxA), or any combination thereof. In some cases, a subject mRNA includes pseudouridine and / or 5-methylcytosine (m5C). One of ordinary skill in the art will understand how to make such an mRNA. For example, in some cases, a subject mRNA (one encoding KLF1, KLF4 or IKZF1) is generated via in vitro transcription (e.g., using an RNA polymerase promoter such as T7 promoter, SP6, and the like). In such cases, pseudouridine, 5-methylcytosine (m5C), N6-methyladenosine (m6A), and / or N'-methyladenosine (m'A) can be present during the transcription reaction so that they are incorporated during transcription. For example, in some cases, pseudouridine is present during the transcription reaction. In some cases, 5-methylcytosine is present during the transcription reaction. In some cases, pseudouridine and 5-methylcytosine are present during the transcription reaction.

[0054] In some cases, a 5’ and / or 3’ untranslated region (UTR) of an mRNA encoding a transcription factor can be changed relative to the natural mRNA encoding the transcription factor. Alternative UTRs can be sourced from another natural regulatory sequence (e.g., a 5’ and / or 3’ UTR from a different mRNA such as a human beta-globin gene) or a synthetic sequence, e g., as designed by a human or computer algorithm. As an example, the 5’ UTR used in the working examples below was SEQ ID NO: 11 and the 3’ UTR used in the working examples below was SEQ ID NO: 12. As such, in some cases, a 5’ UTR (which can be present in an mRNA, and can also be present in a DNA encoding the mRNA) includes the sequence set forth as SEQ ID NO: 11; and in some cases, a 3’ UTR (which can be present in an mRNA, and can also be present in a DNA encoding the mRNA) includes the sequence set forth as SEQ ID NO: 12.

[0055] In some cases, expression of a transcription factor (e g., via regulation of translation and / or regulation of mRNA stability) is controlled using a riboswitch (e.g., by an inducible splicing event), which can be used to regulate translation via changes in structure in response to metabolites. As such, in some cases a subject mRNA includes a riboswitch. In some cases,Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 expression of a transcription factor (KLF1, KLF4, IKZF1) is controlled using a protease (e.g., an endogenous protease) paired with a small molecule protease inhibitor.

[0056] In some cases, an mRNA includes one or more base modifications, or one or more modified internucleotide linkages, or one or more sugar modifications, or any combination thereof. In some embodiments, a nucleic acid (e.g., an mRNA, a guide RNA, and the like) has one or more modifications, e.g., a base modification, a backbone modification, etc., to provide the nucleic acid with a new or enhanced feature (e.g., improved stability). A nucleoside is a basesugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', the 3', or the 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound; however, linear compounds are suitable. In addition, linear compounds may have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

[0057] As used herein, the term “2'-modified” or “2'-substituted” means a sugar comprising a substituent at the 2'-position other than H or OH. 2'-modified nucleotides, include moieties with 2' substituents selected from alkyl, allyl, amino, azido, fluoro, thio, O-alkyl, e.g., O-methyl, O-allyl, OCF3, O-(CH2)2-O-CH3 (e g., 2'-O-methoxyethyl (MOE)), O-(CH2)2SCH3, )-(CH2)2-ONR2, and O-CH2C(O)-NR2, where each R is independently selected from H, alkyl, and substituted alkyl.

[0058] Suitable nucleic acid modifications include, but are not limited to: 2’-O-methyl modified nucleotides, 2’ -fluoro modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphorothioate linkages, and a 5’ cap (e.g., a 7-methylguanylate cap (m7G)). Additional details and additional modifications are described below.Atty Docket No.: NEWL-003 / 01WQ 42005 / 9

[0059] A 2'-O-Methyl modified nucleotide (also referred to as 2'-O-Methyl RNA) is a naturally occurring modification of RNA found in tRNA and other small RNAs that arises as a post-transcriptional modification. Oligonucleotides can be directly synthesized that contain 2'-O-Methyl RNA. This modification increases Tm of RNA:RNA duplexes but results in only small changes in RNA:DNA stability. It is stabile with respect to attack by single-stranded ribonucleases and is typically 5 to 10-fold less susceptible to DNases than DNA. It is commonly used in antisense oligos as a means to increase stability and binding affinity to the target message.

[0060] 2’ Fluoro modified nucleotides (e.g., 2' Fluoro bases) have a fluorine modified ribose which increases binding affinity (Tm) and also confers some relative nuclease resistance when compared to native RNA. These modifications are commonly employed in ribozymes and siRNAs to improve stability in serum or other biological fluids.

[0061] LNA bases have a modification to the ribose backbone that locks the base in the C3'-endo position, which favors RNA A-type helix duplex geometry. This modification significantly increases Tm and is also very nuclease resistant. Multiple LNA insertions can be placed in an oligo at any position except the 3'-end. Applications have been described ranging from antisense oligos to hybridization probes to SNP detection and allele specific PCR. Due to the large increase in Tm conferred by LNAs, they also can cause an increase in primer dimer formation as well as self-hairpin formation. In some cases, the number of LNAs incorporated into a single oligo is 10 bases or less.

[0062] The phosphorothioate (PS) bond (i.e., a phosphorothioate linkage) substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone of a nucleic acid (e.g., an oligo). This modification renders the internucleotide linkage resistant to nuclease degradation. Phosphorothioate bonds can be introduced between the last 3-5 nucleotides at the 5'- or 3'-end of the oligo to inhibit exonuclease degradation. Including phosphorothioate bonds within the oligo (e.g., throughout the entire oligo) can help reduce attack by endonucleases as well.

[0063] In some cases, a nucleic acid (e.g., an mRNA) has one or more nucleotides that are 2'-O-Methyl modified nucleotides. In some cases, a subject nucleic acid (e.g., a nucleic acid, e.g., an mRNA) has one or more 2’-fluoro modified nucleotides. In some cases, a subject nucleic acid (e.g., a nucleic acid, e.g., an mRNA) has one or more LNA bases. In some cases, a subject nucleic acid (e.g., a nucleic acid, e.g., an mRNA) has one or more nucleotides that are linked by a phosphorothioate bond (i.e., the subject nucleic acid has one or more phosphorothioate linkages). In some cases, a subject nucleic acid (e.g., a nucleic acid, e.g., an mRNA) has a 5’ cap (e.g., a 7-Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 methylguanylate cap (m7G)). In some cases, a subject nucleic acid (e.g., a nucleic acid, e.g., an mRNA) has a combination of modified nucleotides. For example, a subject nucleic acid (e.g., a nucleic acid (e.g., a nucleic acid, e.g., an mRNA) can have a 5’ cap (e.g., a 7-methylguanylate cap (m7G)) in addition to having one or more nucleotides with other modifications (e.g., a 2'-O-Methyl nucleotide and / or a 2’ fluoro modified nucleotide and / or a LNA base and / or a phosphorothioate linkage).DNA

[0064] As noted above, in some cases, overexpression is achieved using DNA.

[0065] Suitable nucleic acids comprising nucleotide sequences encoding the one or more transcription factors (KLF1, KLF4, IKZF1, or any combination thereof) and / or a CRISPRa fusion protein include expression vectors, where an expression vector comprising a nucleotide sequence(s) encoding the desired protein(s) is a “recombinant expression vector.” In some embodiments, the recombinant expression vector is a viral construct, e.g., a recombinant adeno-associated virus construct (see, e.g., U.S. Patent No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, etc.

[0066] Suitable expression vectors include, but are not limited to: plasmids; cosmids; minicircles; viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:69166921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector (retrovirus) (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Vims, avian leukosis vims, a lentivims, human immunodeficiency vims, myeloproliferative sarcoma vims, and mammary tumor vims); and the like. Numerous suitable expression vectors are known to those of skill in the art, and many are commercially available. In some cases, a lentivims is used.Atty Docket No.: NEWL-003 / 01WQ 42005 / 9

[0067] Various promoters, including constitutive and inducible promoters, may be used to drive expression (e.g., of KLF1, KLF4, IKZF1, and / or of a CRISPRa fusion protein). The promoter may be a constitutively active promoter, i.e. a promoter is active in the absence externally applied agents, or it may be an inducible promoter (e.g., T7 RNA polymerase promoter, heat shock promoter, Tetracycline-regulated promoter, Steroid-regulated promoter, doxycycline-regulated promoter, mouse metallothionein-I promoter, etc). As used herein, an inducible promoter is a promoter whose activity is regulated upon the application of an agent to the cell, (e.g. doxycycline) or the induced presence of a particular RNA polymerase (e.g., T7 RNA polymerase). In some cases, a lentivirus is used to deliver one or more DNAs that include nucleotide sequence(s) (encoding one or more transcription factors) operably linked to an inducible promoter (e.g., tetracycline-inducible promoter, i.e., tetracycline-regulated promoter, doxycycline-inducible promoter, i.e., doxycycline-regulated promoter, and the like).

[0068] In some cases, a lentivirus is used to deliver one or more DNAs that include nucleotide sequence(s) (encoding one or more transcription factors) operably linked to tetracycline-inducible promoter. In some embodiments, such a lentivirus is produced using HEK293 cells (e.g., HEK293 cells expressing third generation lentiviral packaging vectors).

[0069] In some cases (e.g., in some cases in which a lentivirus is used with a tetracylclineinducible promoter), the promoter is induced for a period of time (e.g., 1-5 days, 1-4 days, 2-5 days, 2-4 days, 3-5 days, 3-4 days, 3 days, 4 days, or 5 days). In such cases, the promoter is induced for 3-5 days. In some such cases, the promoter is induced for a period of time (e.g., 1-5 days, 1-4 days, 2-5 days, 2-4 days, 3-5 days, 3-4 days, 3 days, 4 days, or 5 days), followed by a chase period (e.g., 1-6 days, 1-5 days, 1-4 days, 2-6 days, 2-5 days, 2-4 days, 3-6 days, 3-5 days, 3-4 days, 4-6 days, 4-5 day, 3 days, 4 days, 5 days, or 6 days). In some cases, the promoter is induced for about 4 days, which is followed by a chase period of about 5 days.

[0070] Non-limiting examples of constitutive promoters include those from cytomegalovirus (CMV) immediate early, EFl-a (also known as EFl-a), herpes simplex virus (HSV) thymidine kinase (TK), early and late SV40, long terminal repeats (LTRs) from retrovirus, and the like. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

[0071] An expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression.Atty Docket No.: NEWL-003 / 01WQ 42005 / 9

[0072] In some cases, the nucleic acid introduced into the cell (DNA) will integrate into the genome of the cell. In some such cases, the introduced nucleic acid can include a promoter operably linked to the nucleotide sequence(s) encoding the protein(s) (e.g., KLF1, KLF4, IKZF1, or any combination thereof) such that once integrated, expression of the protein(s) will be under the control of the promoter of the introduced nucleic acid(s). In other cases, the introduced DNA will not include a promoter operably linked to the nucleotide sequence(s) encoding the protein(s) (e.g., KLF1, KLF4, IKZF1, or any combination thereof) - and once integrated, expression of the protein(s) will be under the control of a promoter of the cell’s genome (e.g., based on where the introduced nucleic acid integrated).

[0073] As noted above, in some embodiments, overexpression is achieved by stimulating / inducing expression of the desired protein (e.g., KLF1, KLF4, IKZF1, or any combination thereof) from a promoter (e.g., constitutive, inducible) to which it is operably linked. For example, in some cases, a nucleic acid encoding the protein (e.g., KLF1, KLF4, IKZF1, or any combination thereof) is introduced into the cell, and expression of the protein is under the control of an inducible promoter (i.e., the nucleotide sequence encoding the protein is operably linked to an inducible promoter) - and overexpression is achieved by inducing / stimulating expression from the inducible promoter (e.g., by providing the factor that induces expression). In some embodiments, expression of the desired protein (e.g., KLF1, KLF4, IKZF1, or any combination thereof) is induced / stimulated from its endogenous promoter.CRISPRa fusion proteins (or any RNA guided endonuclease)

[0074] In some cases, overexpression is achieved using a CRISPRa fusion protein (or any RNA guided endonuclease system that can be used in a manner similar to CRISPR systems). Examples of such fusion proteins will be known to one of ordinary skill in the art and any convenient CRISPRa fusion protein can be used. A CRISPRa fusion protein includes a CRISPR / Cas effector protein (e.g., Cas9, Cpfl, etc.) fused to a transcription activating protein.

[0075] Examples of CRISPR / Cas effector proteins will be readily available to one of ordinary skill in the art, and any convenient CRISPR / Cas effector protein can be used. In some embodiments, a subject CRISPR / Cas effector protein will be a Cas9 protein (e.g., Staphylococcus aureus Cas9 (saCas9), Streptococcus pyogenes Cas9 (SpyCas9), Neisseria meningitidis Cas9 (nmCas9), Streptococcus thermophilus Cas9 (stCas9), etc.). In some cases, a subject CRISPR / Cas effector protein will be a Cpfl protein.Atty Docket No.: NEWL-003 / 01WQ 42005 / 9

[0076] In class 2 CRISPR systems, the functions of the effector complex (e.g., the cleavage of target DNA) are carried out by a single protein (which can be referred to as a CRISPR / Cas effector protein) - where the natural protein is an endonuclease (e.g., see Zetsche et al, Cell. 2015 Oct 22;163(3):759-71; Makarova et al, Nat Rev Microbiol. 2015 Nov;13(l l):722-36; Shmakov et al., Mol Cell. 2015 Nov 5;60(3):385-97; Shmakov et al., Nat Rev Microbiol. 2017 Mar; 15(3): 169-182: “Diversity and evolution of class 2 CRISPR-Cas systems”; and Koonin et al., Curr Opin Microbiol. 2017 Jun:37:67-78). As such, the term “class 2 CRISPR / Cas protein” or “CRISPR / Cas effector protein” is used herein to encompass the effector protein from class 2 CRISPR systems - for example, type II CRISPR / Cas proteins (e.g., Cas9), type V CRISPR / Cas proteins (e.g., Cpfl / Casl2a, C2cl / Casl2b, C2C3 / Casl2c, Casl2d / CasY, Casl2e / CasX), and type VI CRISPR / Cas proteins (e.g., C2c2 / Casl3a, C2C7 / Casl3c, C2c6 / Casl3b). Class 2 CRISPR / Cas effector proteins include type II, type V, and type VI CRISPR / Cas proteins, but the term is also meant to encompass any class 2 CRISPR / Cas protein suitable for binding to a corresponding guide RNA and forming a ribonucleoprotein (RNP) complex.

[0077] In some cases, the CRISPR / Cas effector protein (e.g., Cas9) is catalytically inactive (i.e., ‘dead’), which is referred to in the art as a dCas protein (e.g., dCas9). Such a protein will not exhibit the nuclease cleavage activity of the Cas effector protein, but the fusion protein (the CRISPRa fusion protein) will exhibit the activity of the protein to which the dCas protein is fused (i.e., the transcription activating protein).

[0078] A nucleic acid that binds to a CRISPR / Cas effector protein (e.g., a Cas9 protein; a type V or type VI CRISPR / Cas protein; a Cpfl protein; etc.) (and thereby also can bind to a CRISPRa fusion protein) and targets the complex to a specific location within a target nucleic acid (e.g., target DNA) is referred to herein as a “guide RNA” or “CRISPR / Cas guide nucleic acid” or “CRISPR / Cas guide RNA.” It is understood that in some cases a guide nucleic acid includes both RNA and DNA nucleotides (i.e., is a hybrid molecule), but such a hybrid molecule is also referred to herein as a “guide nucleic acid” or “guide RNA.”

[0079] A guide RNA includes a protein-binding region to bind to the CRISPR / Cas effector protein, and also includes a targeting region that provides target specificity to the complex (the RNP complex). The targeting region includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence that is complementary to a target sequence of a target nucleic acid (e.g., target DNA).Atty Docket No.: NEWL-003 / 01WQ 42005 / 9

[0080] A guide RNA can be referred to by the protein to which it corresponds. For example, when the CRISPR / Cas effector protein is a Cas9 protein, the corresponding guide RNA can be referred to as a “Cas9 guide RNA.” Likewise, as another example, when the CRISPR / Cas effector protein is a Cpfl protein, the corresponding guide RNA can be referred to as a “Cpfl guide RNA.”

[0081] In some embodiments, a guide RNA includes two separate nucleic acid molecules: an “activator” and a “targeter” and is referred to herein as a “dual guide RNA”, a “double-molecule guide RNA”, a “two-molecule guide RNA”, or a “dgRNA.” In some embodiments, the guide RNA is one molecule (e.g., for some CRISPR / Cas effector proteins, the corresponding guide RNA is naturally a single molecule; and in some cases, an activator and targeter are artificially covalently linked to one another, e.g., via intervening nucleotides), and the guide RNA is referred to as a “single guide RNA”, a “single-molecule guide RNA,” a “one-molecule guide RNA”, or simply “sgRNA.”

[0082] In some cases, two or more guide RNAs can be used, e.g., to target a CRISPRa fusion protein to more than one target sequence at the same time. For example, in some cases, two or more guide RNAs are used that target different sequences of a targeted promoter or enhancer region (e.g., the promoter region of KLF1, KLF4, IKZF1, or any combination thereof).

[0083] Examples of proteins (or fragments thereof) that can be used to increase transcription (i.e., transcription activating proteins) include but are not limited to: transcriptional activators such as VP 16, VP64, VP48, VP64, VP 160, p65 subdomain (e.g., from NFkB), Rta, VPR (which is a fusion of VP64, p65, and Rta), and activation domain of EDLL and / or TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, and the like; histone lysine demethylases such as JHDM2a / b, UTX, JMJD3, and the like; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, Pl 60, CLOCK, and the like; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, and the like. See, e.g., Chavez et al., Nat Methods. 2015 Apr; 12(4): 326-328. In some cases, the CRISPRa system is a SAM system, which includes 3 components that form the DNA-binding complex: (1) a CRISPRa fusion protein (e.g., dCas9 fused to VP64), (2) MS2 aptamer(s) added to the guide RNA (forming a characteristic stem loop structure recognized by MS2), and (3) transcriptional activators P65 (Nuclear Factor NF -KB p65) and HSF1 (Heat Shock Factor 1) fused with an MS2-tag corresponding to the minimal aptamer-binding peptide of the MS2 coat protein. See, e.g., review articles such as Adli, Nat Commun. 2018 MayAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 15;9(1): 1911; Becirovic, Cell Mol Life Sci. 2022 Feb 12; 79(2): 130; andNidhi S, et al., Int JMol Sci. 2021 Mar 24;22(7):3327.

[0084] A subject CRISPRa fusion protein can include one or more NLSs, and a nucleic acid encoding a subject CRISPRa fusion protein will in some cases be codon optimized (for a desired type of cell, e.g., human cell, CD4 Tn cell, etc.).

[0085] In some embodiments, a nucleic acid encoding the CRISPRa fusion protein is integrated into the genome of a target CD4 Tn cell. In some embodiments, the nucleic acid encoding the CRISPRa fusion protein is extrachromosomal.

[0086] In some cases, the nucleotide sequence encoding the CRISPRa fusion protein is operably linked to a constitutive promoter. In such cases, overexpression of KLF1, KLF4, IKZF1, or any combination thereof occurs in the presence the appropriate guide RNA(s) [e.g., targeting the respective promoter and / or enhancer regions] - as such, overexpression of the one more transcription factors can be initiated by introducing the guide RNA(s). In some cases, the nucleotide sequence encoding the CRISPRa fusion protein is operably linked to an inducible promoter, and overexpression of the one more transcription factors occurs, in the presence the appropriate guide RNA(s) [e.g., targeting the respective promoter(s)], when the appropriate factor is present to induce expression of the CRISPRa fusion protein - as such, overexpression can be initiated by introducing the appropriate factor (e.g., doxycycline) to induce expression of the CRISPRa fusion protein.Delivery: Lipid Nanoparticles (LNPs)

[0087] Methods of introducing nucleic acids (and proteins) into cells will be known to one of ordinary skill in the art, and any convenient method can be used. Such methods include, but are not limited to: lipid nanoparticles (LNPs), infection (e.g., using a virus-like particles (VLPs) or viral transduction, and the like), lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, nucleofection, microinjection, and the like. Such methods can be used to deliver nucleic acids encoding one or more of the subject transcription factors, and can also be used to deliver CRISPR components (e.g., effector protein, nucleic acid encoding same, guide RNA, nucleic acid encoding same).

[0088] In some cases, a nanoparticle is used as a delivery vehicle. As used herein, a “nanoparticle” is a particle having at least one dimension in the range of from 1 nm to 1000 nm,Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 from 20 nm to 750 nm, from 50 nm to 500 nm, including 100 nm to 300 nm, e.g., 120-200 nm. The nanoparticle may have any suitable shape, including, but not limited to, spherical, spheroid, rod-shaped, disk-shaped, pyramid-shaped, cube-shaped, cylinder-shaped, nanohelical-shaped, nanospring-shaped, nanoring-shaped, arrow-shaped, teardrop-shaped, tetrapod-shaped, prismshaped, or any other suitable geometric or non-geometric shape. In certain cases, a nanoparticle includes on its surface one or more targeting moieties, e.g., antibodies, ligands, aptamers, small molecules, etc. Nanoparticles include those described in Wang et al. (2010) Pharmacol. Res.62(2):90-99; Rao et al. (2015) ACS Nano 9(6):5725-5740; and Byrne et al. (2008) Adv. Drug Deliv. Rev. 60(15): 1615-1626.

[0089] In some cases, a lipid nanoparticle (LNP) is used as a delivery vehicle. As used herein, the term “lipid nanoparticle” (LNP) refers to a transfer vehicle (e.g., for delivering a molecular payload such as nucleic acid and / or protein to a cell) comprising one or more lipids (e.g., ionizable lipids, anionic lipids, cationic lipids, non-cationic lipids, neutral lipids, neutral phospholipids, polymerizable lipids, PEG-modified lipids, cholesterol, helper lipids, and the like). Historically, effective LNPs can include, e.g., in some cases, 4 components: an ionizable cationic lipid, zwitterionic phospholipid, cholesterol, and lipid polyethylene glycol) (PEG). The lipid(s) of an LNP can include any convenient lipid. Examples include, but are not limited to DLin-DMA, DLin-K-DMA, 98NI2-5, CI2-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. When an LNP is said to “include” or “comprise” a molecular payload, or cargo, such as a protein or nucleic acid (e.g., mRNA), it is meant herein that the LNP is associated with the payload, such as wherein the LNP is covalently or non-covalently bound to the payload. By way of example, the LNP may encapsulate that payload. In some embodiments, essentially none of the molecular payload present in the formulation is exposed to the environment outside of the LNP (i.e., the payload is fully encapsulated). In some embodiments wherein an LNP comprises a molecular payload, the payload is associated with the LNP but is at least partially exposed to the environment outside of the LNP. By way of illustration, an encapsulation efficiency of 100% refers to an LNP formulation where essentially all the payload is fully encapsulated by the LNP, while an encapsulation rate of 0% refers to an LNP where essentially none of the payload is encapsulated in the LNP, such as with an LNP where the payload is bound to the external surface of the LNP.

[0090] LNP refers to a lipid-based vehicle useful for delivery of nucleic acid molecules and / or proteins and having dimensions on the nanoscale. In some embodiments, the nanoparticle is fromAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 about Inm to about lOOOnm, about lOnm to about 20nm, about 20nm to about 50nm, about 50nm to about 500 nm, or about 50nm to about 200nm (e.g., about 10-500 nm, about 20-500 nm, about 50-400 nm, about 50-300 nm, about 50-200 nm, about 50-150 nm, about 60-120 nm, or about 60-100 nm). As would be known to one of ordinary skill in the art, the average sizes (diameters) of the fully formed LNP, may be measured by dynamic light scattering on a Malvern Zetasizer

[0091] Lipid nanoparticles may be, e.g., microspheres (including unilamellar and multilamellar vesicles, e.g. “liposomes”-lamellar phase lipid bilayers that, in some embodiments are substantially spherical, and can in some cases comprise an aqueous core, e.g., comprising one or more nucleic acids molecules, e.g., one or more RNA molecules), a dispersed phase in an emulsion, micelles or an internal phase in a suspension.

[0092] In some cases, an LNP includes a cationic lipid. Cationic lipids typically have a positively charged head group followed by a hydrophobic tail of varying composition. In an aqueous environment, these cationic lipids form micelles with positively charged surfaces that complex with DNA. Examples of cationic lipids include, but are not limited to: ADC, l,2-dioleoyl-3-dimethylammonium-propane (DODAP), l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOPTAC), l,2-dioleoyl-3-(2-(dimethylamino)ethoxy)propylamine (DODEA), and l,2-dimyristoyl-3-trimethylammonium-propane (DMTAP).

[0093] In some cases, an LNP includes an ionizable lipid. Examples of ionizable lipids include, but are not necessarily limited to: 4A3-SC8, 1014, OF-02, L-319, BP lipid 312, LP01, Lipid HI-45, ALC-0315, lipid A9, D-Lin, DLin-MC3-DMA, ALC-0315, SM-102, LP01, CL1, TCL053, CKK-E12, ATX-002, DLin-DMA, DLenDMA, DLin-D-DMA, DLin-K-DMA, DLin-M-C2-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-KC3-DMA, DLin-KC4-DMA, DLin-C2K-DMA, DLin-MP-DMA, DODMA, 98N12-5, C 12-200, DLin-C-DAP, DLin-DAC, DLinDAP, DLinAP, DLin-EG-DMA, DLin-2-DMAP, KL10, KL22, KL25, Octyl-CLinDMA, Octyl-CLinDMA (2R), Octyl-CLinDMA (2S), and analogs thereof.

[0094] In some cases, an LNP includes a neutral phospholipid. Examples of neutral phospholipids include, but are not necessarily limited to: : 5-heptadecylbenzene-l,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3 -phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 myristoyl-2-palmitoyl phosphatidylcholine (MPPC), l-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), l-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), l-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), l,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoyl phosphatidyl ethanolamine (DMPE). In another embodiment, the neutral phospholipid may be distearoylphosphatidylcholine (DSPC).

[0095] In some cases, an LNP includes a polymerizable lipid, e.g., aPEGylated lipid (e.g., DMG-PEG 2000, DSG-PEG 2000, ALC-0159). PEG-modified lipids (also referred to as PEGylated lipids) include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC 14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified l,2-diacyloxypropan-3-amines. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG (DMG-PEG), PEG-DLPE, PEG DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some embodiments, the PEG-lipid are 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-disteaiyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In some embodiments, the PEG moiety has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In some cases, instead of being PEG-modified, the polymerizable lipid can be modified with other hydrophilic polymers (other than PEG) - e.g., in some cases, the lipids in this paragraph can be modified with a hydrophilic polymer other than PEG.

[0096] In some cases, an LNP includes a molecular payload such as nucleic acid and / or protein (e.g., a CRISPR-Cas effector protein, a DNA or RNA encoding a CRISPR-Cas effector protein, a guide RNA, a DNA encoding a guide RNA, or any combination thereof).

[0097] A variety of different nanoparticles can be employed, including LNPs, polymeric nanoparticles, lipid polymer nanoparticles (LPNP), protein and peptide-based nanoparticles,Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 DNA dendrimers and DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes. (See, e.g., Riley and Vermerris Nanomaterials 2017, 7, 94; Thomas et al., Molecules 2019, 24, 3744; Bochicchio et al., Pharmaceutics 2021, 13, 198; Munagala et al., Cancer Letters 2021, 505, 58; Fu et al., 2020 NanoImpact 20, 100261; and Neshat et al. 2020 Current Opin. Biotechnol. 66:1-10.)

[0098] In some cases, an LNP includes 4A3-SC8, DODAP, DOPE and cholesterol. In some cases, an LNP includes 4A3-SC8, DODAP, DOPE, cholesterol, and DMG-PEG 2000. In some such cases these lipids are used at a ratio of about (5-10):(10-20):(45-55):(50-60):(30-40), e.g., about 7:14:51:53:34 (e.g., 6.7:14.3:50.7:52.7:34.2). When mixing with a nucleic acid (e.g., mRNA) for delivery, in some cases the LNPs are prepared using a ratio of about 1:1 to 5:1 mRNAlipid mixture. For example, in some cases the LNPs are prepared using a ratio of about 3: 1 mRNA:lipid mixture. For example, an LNP can be prepared by mixing (e.g., pipette mixing, microfluidic mixing, and the like) of an aqueous phase mRNA solution and an organic phase lipid solution.

[0099] When delivering LNPs in vivo, any convenient dose can be used. In some embodiments, the dose range is in a range of from 0.05-0.8 mg / kg (e.g., 0.05-0.7, 0.05-0.6, 0.05-0.5, 0.05-0.4, 0.05-0.3, 0.08-0.8, 0.08-0.7, 0.08-0.6, 0.08-0.5, 0.08-0.4, 0.08-0.3, 0.09-0.8, 0.09-0.7, 0.09-0.6, 0.09-0.5, 0.09-0.4, 0.09-0.3, 0.1-0.8, 0.1-0.7, 0.1-0.6, 0.1-0.5, 0.1-0.4, or 0.1-0.3 mg / kg). In some embodiments, the dose is in a range of from 0.1-0.3 mg / kg.Delivery: viruses or virus-like particles (VLPs)

[0100] In some cases, a virus or VLP is used as a delivery vehicle. See above for examples of viral vectors that can be used (e.g., viral vectors base on vaccinia virus; poliovirus; adenovirus; adeno-associated virus (AAV); SV40; herpes simplex virus; human immunodeficiency virus; retrovirus (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0101] Virus-like particles (VLPs) (also known as enveloped delivery vehicles (EDVs)) can also be used to deliver nucleic acids. VLPs have become important tools in biology and medicine. As would be understood by one of ordinary skill in the art, one unique feature of viruses is that their structural proteins, envelope proteins, or capsid proteins, along with other structural proteins, can, either independently or collectively, spontaneously self-assemble to form virus-like particlesAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 (VLPs) without the viral genome. Thus, any virus can be utilized to develop VLPs; VLPs can be developed, e.g., by cloning the structural genes that code for the proteins of a virus of interest into an expression vector. The expression vector depends on the expression system in which the protein(s) will be expressed and, sometimes, the protein-coding sequence is codon-optimized if the expression system (including that of mammalian, insects, and bacteria) is different to the cells which the virus of interest will infect (e g., CD4 Tn cells). The vector harboring the DNA of the structural protein(s) can then be transfected / transformed into cells of interest, where the DNA is transcribed and translated. Translated proteins fold and assemble to form VLPs. VLPs have many applications in biomedical sciences, for example, but not limited to: (i) therapy — the delivery of drugs / cargo to target cells; (ii) in vivo imaging — VLPs loaded with fluorophores; (iii) diagnostic tests — utilizing armored RNA as positive controls for infectious diseases; and iv) vaccine development. For information related to VLPs, please see, e.g., Mohsen et al., Cell Mol Immunol 19, 993-1011 (2022); Kheirvari et al., Viruses. 2023 May 2; 15(5): 1109; Mejia-Mendez et al., Int J Mol Sci. 2022 Aug 2;23(15) : 8579; and Hamilton et al., Nat Biotechnol 42, 1684-1692 (2024)).Pharmaceutical Compositions

[0102] Also provided are pharmaceutical compositions useful for delivering a molecular payload to an individual according to the methods of the disclosure as described herein. Further, the present disclosure provides dosing regimens for administering the described pharmaceutical compositions. For example, the present disclosure provides pharmaceutical compositions comprising: a) one or more nucleic acids encoding the one or more transcription factors (e.g., mRNA(s), DNA(s), and in some cases a delivery vehicle such as an LNP or virus or VLP); and b) a pharmaceutically acceptable carrier, diluent, excipient, or buffer. As another example, the present disclosure provides pharmaceutical compositions comprising: a) a CRISPRa fusion protein and / or a nucleic acid encoding the CRISPRa fusion protein, and / or one or more guide RNAs or nucleic acid(s) encoding the one or more guide RNAs (and in some cases a delivery vehicle such as an LNP or virus or VLP); and b) a pharmaceutically acceptable carrier, diluent, excipient, or buffer. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for use in a human. See elsewhere herein for additional disclosure related to Pharmaceutical Compositions.Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 Additional details related to methods

[0103] The methods can utilize any of the compositions described herein. Likewise, it is understood that any of the compositions described herein in the context of a method are also considered disclosed in the context of a composition.

[0104] The present disclosure provides methods of overexpressing one or more transcription factors (KLF1, KLF4, IKZF1, or any combination thereof) in a CD4 Tn cell (e.g., in a population of CD4 Tn cells). The CD4 Tn cell(s) can be in vitro (e.g., in culture - in some cases and established cell line and in some cases an ex vivo cell recently isolated from in individual), ex vivo (e.g., recently isolated from an individual, or in vivo).

[0105] In some embodiments, CD4 Tn cells are stimulated / activated via CD3 and / or CD28 prior to overexpression of the one or more transcription factors. For example, CD4 Tn cells can be activated via CD3 using an anti-CD3 antibody (such as a plate bound anti-CD3 antibody) prior to overexpression. In some cases, CD4 Tn cells are activated via CD3 using an anti-CD3 antibody (such as a plate bound anti-CD3 antibody), and activated via CD28 using an anti-CD28 antibody (such as a soluble co-stimulatory antibody against CD28) prior to overexpression.

[0106] In some cases, a subject method results in reversion of an age-related defect / dysfunction (e.g., accelerated T cell differentiation). In some cases, a subject method results in reduction of accelerated T cell differentiation in a CD4 Tn cell, stimulated (induced) youthful gene expression in a CD4 Tn cell, and / or reduced cellular age of a CD4 Tn cell.

[0107] In some embodiments, the compositions and methods are for use in vivo (i.e., in an individual, e.g., as a method of treatment). As such, in some embodiments, a subject method results in reducing accelerated T cell differentiation in a CD4 Tn cell in vivo, stimulated (induced) youthful gene expression in a CD4 Tn cell in vivo, and / or reduced cellular age in a CD4 Tn cell in vivo.

[0108] Youthful gene expression refers to a gene expression profile that resembles (is more similar to) a young cell (CD4 Tn cell) as compared to an old cell (CD4 Tn cell). For such purposes, in some cases, a young CD4 Tn cell is a CD4 Tn cell that is from an individual who is 29 years old or younger, and an old CD4 Tn cell is a CD4 Tn cell this is from an individual who is 61 years old or older. Such an expression profde can be determined using any convenient method (e.g., using microarrays, using nucleic acid sequencing, e.g., using high throughput / next-generation sequencing methods). Gene expression profiles can be determined for cells in culture, and can also be determined for cells in vivo, e.g., via biopsy. Surrogate markers for determining whetherAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 youthful gene expression has been achieved may include, e.g., measuring CD45RA protein level (see below). Additional surrogate markers for determining whether youthful gene expression has been achieved may include, e.g., measuring production of inflammatory cytokines (youthful CD4 Tn cells produce less inflammatory cytokines over time), measuring proliferation (youthful CD4 Tn cells proliferate over a longer time frame under chronic stimulation), and evaluating polarization (youthful CD4 Tn cells polarize more readily across Thl, Th2, and Thl7 states).

[0109] In some embodiments, a cellular age can be determined by assessing gene expression (e.g., using a machine learning algorithm). For example, gene expression profiles from young and old cells can be obtained, and a machine learning algorithm can be trained to distinguish old from young cells, or, e.g., to determine a ‘cellular age’ or ‘age score’ for a given cell (e.g., CD4 Tn cell). As such, in some cases, cell age is determined after overexpression of the subject transcription factor(s). In some cases, a subject method induces (stimulates) a more youthful gene expression profile (i.e., more youthful gene expression) (e.g., compared to a control). In some cases, a subject method induces (stimulates) youthful gene expression. In some cases, a subject method causes a decrease in cellular age (reduced cellular age). In some cases, a subject method results in a cellular age of 29 years old or younger. In general, young cells have improved function relative to old cells.

[0110] In some cases, a subject method includes a step of measuring (e.g., assaying for a particular feature, e.g., cell function, youthful gene expression, reduced cellular age, marker expression, and the like). For example, reduced expression of CD45RA can be an indicator of aging CD4 T cells, e.g., an indicator of accelerated T cell differentiation. Accelerated T cell differentiation has been implicated in the progression of rheumatoid arthritis [see, e.g., Weyand et al., Nat Rev Rheumatol. 2017 May;13(5):291-301; Weyand et al., Free Radic Biol Med. 2018 Sep;125:36-43; and Wu et al., Immunometabolism. 2020;2(2):e200017)], and the presence of less differentiated T cells subsets improves outcomes in both infectious disease and oncology [see, e.g., Alizadeh et al., Cancer Immunol Res. 2019 May;7(5):759-772; Fraietta et al., Nat Med. 2018 May;24(5):563-571; and Ribeiro et al., J Virol. 2014 Dec;88(23): 13836-44],

[0111] As noted above, additional examples of ways to measure increased CD4 Tn cell function include, but are not limited to: measuring production of inflammatory cytokines (youthful CD4 Tn cells (increased function) produce less inflammatory cytokines over time), measuring proliferation (youthful CD4 Tn cells (increased function) proliferate over a longer time frameAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 under chronic stimulation), and evaluating polarization (youthful CD4 Tn cells (increased function) polarize more readily across Thl, Th2, and Th 17 states).

[0112] As an example, in the working examples below, CD4 T cells from aged donors exhibited a marked downregulation of CD45RA protein expression after 10 days in culture while young donors exhibited a significantly higher expression level of CD45RA. As such, in some cases, a subject method results in increased CD45RA expression. In some cases, a subject method (e.g., any of the methods disclosed herein) includes measuring CD45RA expression (e.g., using and antiCD45RA antibody such as CD45RA-PE / Cy7 from Biolegend (304126)) in cells after and / or before overexpression of the one or more transcription factors. The measured level can be compared to a control (e.g., CD45RA levels measured in parallel from comparable cells (e.g., mock treated cells) without overexpression, or predetermined levels such as those previously measured for comparable cells with and / or without overexpression of the subject one or more transcription factors).

[0113] In some cases, overexpression of one or more of the subject transcription factors results in CD45RA levels that are increased by 5% or more (e.g., 10% or more, 15% or more, 20% or more, 30% or more, 50% or more, 60% or more, 80% or more, or 100% or more) relative to a control. In some cases, CD45RA levels after overexpression is initiated is 1.1 times or more (e.g., 1.2 times or more, 1.3 times or more, 1.5 times or more, 1.6 times or more, 1.8 times or more, 2 times or more, 3 times or more, 5 times or more) that of the CD45RA levels of a control (e.g., CD45RA levels prior to overexpression of a subject transcription factor(s), CD45RA levels of a comparable cell(s) in which overexpression is not performed, an appropriate predetermined value, and the like).

[0114] In some cases, a cellular function of interest is increased by 5% or more (e.g., 10% or more, 15% or more, 20% or more, 30% or more, 50% or more, 60% or more, 80% or more, 100% or more) relative to an appropriate control. In some cases, a cellular function of interest after overexpression is initiated is 1.1 times or more (e.g., 1.2 times or more, 1.3 times or more, 1.5 times or more, 1.6 times or more, 1.8 times or more, 2 times or more, 3 times or more, 5 times or more) that of the cellular function of interest of a control.

[0115] As noted above, overexpression can be achieved in a number of different ways (e.g., introduction of nucleic acid(s) encoding the transcription factor(s), using CRISPRa to induce gene expression, and the like). In some cases, an LNP is used to deliver the molecular payload (e.g.,Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 one more DNAs, one or more mRNAs, one or more guide RNAs, one or more proteins, e.g., a CRISPRa fusion protein). In some cases, a virus or VLP is used to deliver the molecular payload.

[0116] CD4 Tn cells (in which the one or transcription factors are overexpressed) can be human or non-human, especially mammalian, including, but not limited to, human and non-human primate cells (including simian and human); mammalian sport animal cells (e.g., horses); mammalian farm animal cells (e.g., sheep, goats, llama, camel, pig, cow, etc ); mammalian pet cells (e.g., dogs, cats, etc.); and rodent cells (e.g., mice, rats, etc.). In some cases, the CD4 Tn cell is human. In some cases the CD4 Tn cell(s) (the targeted cell(s)) is from an individual who is (or was) 61 years of age or older at the time the cell(s) was obtained.

[0117] In some cases, a subject method is a method of treatment. For example, in some cases, a subject method includes overexpressing one or more of the above transcription factors in CD4 Tn cells of an individual, e.g., via introducing a CRISPRa system to stimulate expression or via introducing nucleic acid(s) encoding the transcription factor(s). In some cases, a subject method includes overexpressing one or more of the above transcription factors in CD4 Tn cells outside of an individual (e.g., in a cell ex vivo, in cell culture in vitro), and then transplanting (administering) the CD4 Tn cells into an individual. In some cases, the CD4 Tn cells (now overexpressing the one or more transcription factors) are derived from the same individual from whom they (CD4 Tn cells prior to overexpression) were isolated. For example, a CD4 Tn cell(s) can be harvested from the individual, a nucleic acid molecule (encoding the one or more transcription factors) can be introduced into the harvested cell(s) ex vivo, and the CD4 Tn cell(s) can then be introduced back into the same individual, e.g., in some cases after culture of the cell(s) to generate a (larger) population. As such, in some cases, the CD4 Tn cell(s) will be autologous to the individual. In some cases, the CD4 Tn cell(s) will be allogeneic to the individual. For information related to cell transplantation, see elsewhere herein, as well as, e.g., Falkenburg et al., Biol Blood Marrow Transplant. 2008 Jan;14(l Suppl 1):136-41; Goldsmith et al., Front Oncol. 2020 Dec 22; 10:608916; Wu et al., Clin Transl Med. 2025 Apr;15(4):e70306; Goetzl et al., Am J Med. 2025 Mar 27:S0002-9343(25)00194-9; Deng et al., Ther Adv Hematol. 2025 Mar 25; 16:20406207251326802.

[0118] The term “treating” or “treatment” as used herein means the treating or treatment of a disease, disorder, or medical condition in a patient, such as a mammal (particularly a human) that includes: (a) preventing the disease, disorder, or medical condition (or associated symptoms) from occurring, such as, prophylactic treatment of a subject; (b) ameliorating the disease, disorder, orAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 medical condition (or associated symptoms), such as, eliminating or causing regression of the disease, disorder, or medical condition in a patient; (c) suppressing the disease, disorder, or medical condition (or associated symptoms), for example by, slowing or arresting the development of the disease, disorder, or medical condition in a patient. In some cases, a treatment alleviates a symptom(s) of the disease, disorder, or medical condition in a patient.

[0119] “Pharmaceutically effective amount” and “therapeutically effective amount” or simply “effective amount” refer to an amount of a compound sufficient to treat an injury, disorder, or disease or one or more of its symptoms and / or to prevent the occurrence of the disease or disorder. In reference to CD4 Tn cells, e.g., in the context of a disease or disorder, a pharmaceutically or therapeutically effective amount comprises an amount sufficient to, e.g., reduce accelerated T cell differentiation, induce youthful gene expression, and / or reduce cellular age.

[0120] A subject agent can be formulated with a pharmaceutically acceptable carrier (one or more organic or inorganic ingredients, natural or synthetic, with which a subject agent is combined to facilitate its application). A suitable carrier can include sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art.

[0121] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro, (2000) Remington: The Science and Practice of Pharmacy, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7. sup. th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3. sup. rd ed. Amer. Pharmaceutical Assoc.

[0122] Such compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents,Atty Docket No.: NEWL-003 / 01WO 42005 / 9 compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and nonaqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions.

[0123] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as set forth herein or known to one of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.

[0124] Compositions suitable for parenteral administration comprise aqueous and non-aqueous solutions, suspensions or emulsions of the active compound. Preparations are typically sterile and can be isotonic with the blood of the intended recipient. Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal, vegetable or synthetic oils.

[0125] Cosolvents and adjuvants may be added to the formulation. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Adjuvants include, for example, surfactants such as, soya lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.

[0126] Pharmaceutical compositions and delivery systems appropriate for subject agents and methods and uses of are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20. sup. th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18. sup. th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12. sup. th ed., Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) ll.sup.th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp.253-315).

[0127] The terms “patient”, “individual”, and “subject” refer to human and non-human subjects, especially mammalian subjects, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses); mammalian farm animalsAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). In some cases, the individual is a human.

[0128] Indications: In some cases, the individual has a has an infectious disease, an autoimmune or inflammatory disease or disorder, and / or a hyperproliferative condition, such as a cancer. For example, in some cases, the individual has rheumatoid arthritis, a solid tumor cancer, chronic lymphocytic leukemia (CLL), a viral infection (e.g., a chronic HIV-1 Infection), a bacterial infection, or any combination thereof.

[0129] As used herein “cancer” includes any form of cancer, e.g., solid tumor cancers (e.g., liver, lung, prostate, breast, bladder, colon, ovarian, pancreas, kidney, glioblastoma, medulloblastoma, leiomyosarcoma, head & neck squamous cell carcinomas, melanomas; etc.), liquid cancers, leukemia, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), lymphomas, mesothelioma (MSTO), including both primary and metastatic tumors, and the like. In some cases, the individual has recently undergone treatment for cancer (e.g., radiation therapy, chemotherapy, surgical resection, etc.) and are therefore at risk for recurrence. Any and all cancers are suitable cancers to be treated / targeted by the subject methods, compositions, and kits. In some cases the cancer is liver cancer, bile duct cancer, bladder cancer, brain cancer, cancer of the nervous system, breast cancer, cervical cancer, colon cancer, esophagus cancer, head and neck cancer, renal cancer, cancer of the large intestine, leukemia, lymphoma, lung cancer, melanoma, skin cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular cancer, or cancer of the uterus. In some cases, the cancer is liver cancer, lung cancer, ovarian cancer, prostate cancer, colorectal cancer, renal cancer, bladder cancer, breast cancer, thyroid cancer, pleural cancer, pancreatic cancer, uterine cancer, cervical cancer, testicular cancer, anal cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gall bladder cancer, appendix cancer, small intestine cancer, gastric cancer, cancer of the central nervous system, skin cancer, head and neck cancer, or blood cancer.

[0130] In particular embodiments, the present disclosure provides compositions and methods for treating a proliferative disorder. Such disorders include, without limitation, hyperproliferative skin disorders. Hyperproliferative skin disorders include benign conditions, such as psoriasis and malignant conditions, such as melanoma. Particular hyperproliferative skin disorders also include non-melanoma skin cancers, such as basal cell carcinoma and squamous cell carcinoma, and pyogenic granuloma. In another aspect, proliferative disorders treated as described herein include cancers and hematopoietic disorders, including leukemias, lymphomas, myeloproliferativeAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 neoplasms and myelodysplastic syndromes. By way of example, hematopoietic disorders treated as described herein include, without limitation, Hodgkin's and non-Hodgkin’s lymphoma, T — cell lymphoma, primary cutaneous T-cell lymphoma, mycosis fungoides, Sezary syndrome, chronic lymphocytic leukemia (CLL), smoldering or indolent multiple myeloma, acute myeloid leukemia (AML), anaplastic large cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated DLBCL (ABC DLBCL), chronic lymphocytic lymphoma, primary effusion lymphoma, Burkitt lymphoma / leukemia, acute lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, polycythemia vera, Waldenstrom's macroglobulinemia, splenic marginal zone lymphoma, multiple myeloma, plasmacytoma, and intravascular large B-cell lymphoma.

[0131] In one aspect the present disclosure provides compositions and methods for treating autoimmune and inflammatory disorders. Such disorders include those in which a subject's immune system has an aberrant or pathological immune or inflammatory response. Examples of such autoimmune and inflammatory disorders that may be treated with a compound, pharmaceutical composition, or method described herein include, but are not limited to: acne vulgaris, acute disseminated encephalomyelitis, acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, Aicardi-Goutieres syndrome (AGS), alopecia areata, alopecia totalis, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, asthma, atopic dermatitis, autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease, autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura, autoimmune thyroid disease, autoimmune urticaria, axonal or neuronal neuropathies, balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas disease, chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), chronic active hepatitis, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal ostomyelitis, Churg-Strauss syndrome, cicatricial pemphigoid / benign mucosal pemphigoid, Crohn's disease, Cogans syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST disease, Cushing's disease, cytokine release syndrome, demyelinating neuropathies, depression, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressier's syndrome, dry eye syndrome DESAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 (keratoconjunctivitis sicca), endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, graft-versus-host disease (GVDH), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch- Schonl ein purpura, herpes gestationis, hidradenitis suppurativa, hypogammaglobulinemia, idiopathic thrombocytopenic purpura, IgA nephropathy, IgG4-related sclerosing disease, inflammatory bowel disease (IBD), immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes (Type 1 diabetes), juvenile dermatomyositis (JDM), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease, lupus, lupus nephritis, Lyme disease, chronic, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis (MS), myasthenia gravis, myositis, narcolepsy, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, pediatric autoimmune neuropsychiatric disorders associated with streptococcus, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria p, Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polycystic ovary syndrome (PCOS), Type I, II, & III autoimmune polyglandular syndromes, polymyalgia rheumatica, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, plaque psoriasis, pustular psoriasis, palmoplantar pustulosis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud’s phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, Schnitzler’s syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm & testicular autoimmunity, stiff person syndrome, stimulator of interferon genes (STING)-associated vasculopathy with onset during infancy (SAVI), subacute bacterial endocarditis, Susac's syndrome, sympathetic ophthalmia, systemic lupus erythematosus (SLE), Takayasu's arteritis, temporal arteritis / Giant cell arteritis, thrombocytopenic purpura, Tolosa-Hunt syndrome, transplant rejection (allograftAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 transplant rejection), transverse myelitis, Type 1 diabetes, ulcerative colitis, undifferentiated connective tissue disease, uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener's granulomatosis. In some cases, the autoimmune disease or disorder is rheumatoid arthritis.

[0132] In particular embodiments, the present disclosure provides methods and compositions for treating an infectious disease. The term “infectious disease” may collectively refer to infectious diseases caused by organisms such as bacteria, viruses, fungi, protozoa, parasites, and the like. For example, in some cases an infectious disease is a viral infection caused by, e.g., Human Immunodeficiency Virus (HIV), Hepatitis C virus (HCV), Hepatitis B virus (HBV), Epstein-Barr virus (EBV), Human Herpes Virus (HHV), Influenza A virus (IAV), a coronavirus (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV), cytomegalovirus (CMV, encephalomyocarditis virus (EMCV), and the like. In some cases, the infectious disease is HIV.

[0133] Administration routes: Administration of compounds of the present disclosure (e.g., nucleic acid(s), protein(s), cell (e.g., T cells), or delivery vehicles such as LNPs, viruses, or VLPs), can be achieved using any convenient route. In some cases, administration is intravenous, intraperitoneal, intratumoral, or subcutaneous. For example, compounds of the present disclosure can be administered to a subject in any suitable form, e.g., in the form of a pharmaceutically acceptable composition, and can be formulated for any convenient route of administration, e.g., can be administered systemically or locally, and by any convenient route, e.g., oral, topical or parenteral administration. Routes of administration can include, but are not necessarily limited to: oral (alimentary), intravenous, subcutaneous, intramuscular, intraperitoneal, intratumoral, mucosal, peritumoral, intra-pleural, intracranial (e.g., convection enhanced delivery), intraocular, topical, dermal, intradermal, transdermal, transmucosal, retro-orbital (e.g., retro-orbital injection), respiration, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavity, iontophoretic, intraocular, ophthalmic, optical, intraglandular, intraorgan, and intralymphatic, etc. In some cases, administering to a subject is via local administration (e.g., injection). In some cases, administration is systemic administration (e.g., intravenous). Where a compound is provided as a liquid injectable (such as in those embodiments where the compound is administered intravenously or directly into a tissue), the compound can be provided as a ready-to-use dosage form, or as a reconstitutable storage- stable powder or liquid composed of pharmaceutically acceptable carriers and excipients.

[0134] In some cases, LNPs (e.g., those with mRNA(s) encoding the one or more transcription factors) are delivered via intravenous, intraperitoneal, or subcutaneous injection. In some cases,Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 LNPs (e.g., those with mRNA(s) encoding the one or more transcription factors) are delivered via intravenous injection.

[0135] Methods for formulating compounds can be adapted from those readily available. For example, compounds can be provided in a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present disclosure and a pharmaceutically acceptable carrier. The pharmaceutical composition may optionally include other additives (e g., buffers, stabilizers, preservatives, and the like). In some embodiments, the formulations are suitable for administration to a mammal (e.g., a rodent such as a mouse or a rat, a pig, a horse, a rabbit, a dog, a cat, a non-human primate, and the like), such as those that are suitable for administration to a human.

[0136] A subject agent (e.g., a subject nucleic acid, a subject cell, and the like) can be formulated with a pharmaceutically acceptable carrier (one or more organic or inorganic ingredients, natural or synthetic, with which a subject agent is combined to facilitate its application). A suitable carrier can include sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art.

[0137] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro, (2000) Remington: The Science and Practice of Pharmacy, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7. sup. th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3. sup. rd ed. Amer. Pharmaceutical Assoc.

[0138] Such compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents,Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and nonaqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions.

[0139] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as set forth herein or known to one of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.

[0140] In certain embodiments, the compositions may include CD4 Tn cells present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCl, MgCh, KC1, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc ), a nuclease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions.

[0141] A population may include a therapeutically effective amount of cells. By “therapeutically effective amount” it is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to affect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease or disorder associated, e.g., with the target cell or a population thereof, as compared to a control. An effective amount can be administered in one or more administrations.

[0142] A “therapeutically effective amount” of such cells may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the cells to elicit a desired response in the subject. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician inAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). In certain embodiments, a therapeutically effective amount of spreader cells (e.g., T cells) may be 100,000-50M of the cells. However, in other embodiments, a therapeutically effective amount of spreader cells (e.g., T cells) may be 5OM-5OOM cells.

[0143] Cells that can be administered to a subject as described herein include CD4 Tn cell having a subject nucleic acid and / or overexpressing one or more transcription factors (KLF1, KLF4, IKZF 1 , or any combination thereof). In one aspect, such cells can be a T cell engineered to express a T cell receptor of interest (such a cell is referred to herein as a “TCR-T cell”) (autologous or allogeneic). As such, compositions described herein (e.g., one or more nucleic acids encoding the one or more transcription factors, e.g., plasmid(s), viral vector(s), LNPs comprising, and the like)) can be used as manufacturing agents for the production of CAR-T cells and / or TCR-T cells.

[0144] For example, disclosed herein are methods (for producing CAR-T cells and / or TCR-T cells (autologous or allogeneic)) in which the CD4 Tn cell in which overexpression occurs is a CAR-T cell or a TCR-T cell. For example, in some cases, a subject method includes overexpressing one or more transcription factors in a CD4 Tn cell (e.g., to reduce accelerated T cell differentiation, to induce youthful gene expression, to reduce cellular age, and the like), where the one or more transcription factors comprise: KLF1, KLF4, IKZF1, or any combination thereof, and where the CD4 Tn cell is a CAR-T cell. In some cases, a subject method includes overexpressing one or more transcription factors in a CD4 Tn cell (e.g., to reduce accelerated T cell differentiation, to induce youthful gene expression, to reduce cellular age, and the like), where the one or more transcription factors comprise: KLF1, KLF4, IKZF1, or any combination thereof, and where the CD4 Tn cell is a TCR-T cell.

[0145] In some cases, the CD4 Tn cell can be a CAR-T cell or a TCR-T cell prior to the overexpressing (meaning, the overexpression of the one or more transcription factors can be in cells that are already TCR-T cells or CAR-T cells prior to the overexpressing). In some cases, the CD4 Tn cells can be converted into a CAR-T cell or a TCR-T cell after said overexpressing (meaning that after initiating overexpression of the one or more transcription factors, the desired receptor (CAR-T or TCR-T) can then be expressed).

[0146] The cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the cells of the present disclosure can be formulated for administration by combination with appropriate excipients, diluents and / or the like.Atty Docket No.: NEWL-003 / 01WQ 42005 / 9

[0147] Formulations of the cells suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.

[0148] The cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration. In some embodiments, cells containing the vector may be administered locally to the brain to a brain tumor, e.g., intracranially or intratumorally (e.g., by direct injection into the brain cavity or tumor).

[0149] An aqueous formulation of the cells may be prepared in a pH-buffered solution, e.g., at a pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.

[0150] A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.

[0151] In some embodiments, a composition includes cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w / v).Co-Administration

[0152] In some cases, any of the compositions disclosed herein, i.e., a subject agent (e.g., one or more nucleic acids, such as mRNAs, encoding KLF1, KLF4, IKZF1 or any combination thereof,Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 CD4 Tn cells that include said nucleic acids, or any combination thereof) can be administered with an additional therapy or agent (e.g., an agent or therapy used to treat an infectious disease, an autoimmune disease or disorder, or a cancer).

[0153] In some cases, the additional therapy or agent is engineered CAR-T cell therapy. As one example, a CD4 Tn cell having a subject nucleic acid and / or overexpressing one or more transcription factors (KLF1, KLF4, IKZF1, or any combination thereof) can be an engineered CAR-T cell (autologous or allogeneic). Likewise, a CD4 Tn cell having a subject nucleic acid and / or overexpressing one or more transcription factors (KLF1, KLF4, IKZF1, or any combination thereof) can be a T cell engineered to express a T cell receptor of interest (such a cell is referred to herein as a “TCR-T cell”) (autologous or allogeneic). As such, compositions described herein (e.g., one or more nucleic acids encoding the one or more transcription factors, e.g., plasmid(s), viral vector(s), LNPs comprising, and the like)) can be used as manufacturing agents for the production of CAR-T cells and / or TCR-T cells.

[0154] As another example of co-administration, CD4 Tn cells having a subject nucleic acid and / or overexpressing one or more transcription factors (KLF1, KLF4, IKZF1, or any combination thereof) can be co-administered with engineered CAR-T cells and / or with TCR-T cells.

[0155] The terms "co-administration", “co-administer”, and "in combination with" include the administration of two or more therapies either simultaneously, concurrently or sequentially within no specific time limits. In some embodiments, agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect at the same time. In some embodiments, therapeutic agents are in the same composition or unit dosage form. In other embodiments, therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first therapy (e.g., agent) can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy (e.g., agent).Atty Docket No.: NEWL-003 / 01WQ 42005 / 9

[0156] A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present disclosure.

[0157] Treatment with a subject agent can be combined with another therapy such as chemotherapy, radiotherapy, and / or other therapies such as immunotherapies to enhance effect. In some embodiments, a second treatment includes a second agent - and the second agent is a cancer targeting agent specifically binds a cancer cell antigen.

[0158] Examples of second agents include but are not limited to: antiviral agents (e.g., acyclovir, gancyclovir), antimicrobial agents, antiprotozoal agents, antifungal agents, antibiotics (e.g., penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, and the like), anti-autoimmune agents [e.g., glucocorticoids, cytostatic agents (purine analog), alkylating agents (cyclophosphamide, nitrosoureas, and platinum compounds), anti-metabolites (methotrexate, azathioprine, and mercaptopurine), cytotoxic antibiotics (dactiomycin anthracyclines, mitomycine C, blemocyin, and mithramycin), antibodies (anti-CD20, anti-CD-25, anti-CD3 (OTK3), monoclonal antibodies, ATGAM® and thymoglobulin), cyclosporin, tacrolimus, rapamycine (siolimus), interferons (e.g., IFN- ), TNF binding proteins (infiximab, etanercept, and adalimumab), mycophenolate, fingolimod, myriocin (thermozymocidin), and the like], Atezolizumab, Avastin (Bevacizumab), Bevacizumab, Cabometyx (Cabozantinib-S-Malate), Cabozantinib-S-Malate, Cyramza (Ramucirumab), Infigratinib Phosphate, Keytruda (Pembrolizumab), Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Nexavar (Sorafenib Tosylate), Nivolumab, Opdivo (Nivolumab), Pemazyre (Pemigatinib), Pembrolizumab, Pemigatinib, Ramucirumab, Regorafenib, Sorafenib Tosylate, Stivarga (Regorafenib), Tecentriq (Atezolizumab), and Truseltiq (Infigratinib Phosphate).

[0159] In some embodiments, a second treatment is a cancer therapy. For example, in some cases, the second treatment is chemotherapy (i.e., chemotherapy is administered to the individual). In some cases, the second treatment is radiotherapy (i.e., radiotherapy is administered to the individual).

[0160] Co-administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the drug / antibody / agent with respect to the administration of an agent or agents of this disclosure. In some cases, a subject agent is formulated with one or more agents that potentiate activity, or that otherwise increase the therapeutic effect (such as an immunomodulatory agent, a tumor-directed antibody, and the like).Atty Docket No.: NEWL-003 / 01WQ 42005 / 9

[0161] In some cases, a subject agent is used in a combination therapy (is co-administered) with a cancer targeting agent (e.g., an agent that specifically binds a cancer antigen, e.g., a cell-specific antibody selective for a tumor cell marker). Any convenient cancer cell targeting agent can be used. In some cases, the cancer cell targeting agent is a specific binding agent (e.g., a polypeptide such as an antibody that includes an antigen binding region specific for a cancer antigen) that specifically binds a cancer antigen of cancer cells (e.g., CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD47, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD274 (PD-L1), EpCam, EGFR, 17-1 A, HER2, CD117, C-Met, PTHR2, HAVCR2 (TIM3), and SIRPA). As such, in some cases, a subject method includes co-administering a subject agent and a cancer cell targeting agent that is a specific binding agent (e.g., a polypeptide such as an antibody that includes an antigen binding region specific for a cancer antigen) that specifically binds an antigen (e.g., a cancer antigen) selected from: CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD47, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD274 (PD-L1), EpCam, EGFR, 17-1A, HER2, CD117, C-Met, PTHR2, HAVCR2 (TIM3), and SIRPA.

[0162] In some cases, a subject agent is used in a combination therapy (is co-administered) with: cetuximab (binds EGFR), panitumumab (binds EGFR), rituximab (binds CD20), trastuzumab (binds HER2), pertuzumab (binds HER2), alemtuzumab (binds CD52), brentuximab (binds CD30), tositumomab, ibritumomab, gemtuzumab, ibritumomab, or edrecolomab (binds 17-1A), or any combination thereof

[0163] In some cases, a subject agent is used in a combination therapy (is co-administered) with a cancer drug such as: carboplatin, cisplatin, docetaxel (taxotere), gemcitabine (gemzar), nab-paclitaxel (abraxane), paclitaxel (taxol), pemetrexed (alimta), or vinorelbine (navelbine), or any combination thereof

[0164] In some cases, a subject agent is used in a combination therapy (is co-administered) with a cancer drug such as: Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), Afatinib Dimaleate, Afinitor (Everolimus), Afinitor Disperz (Everolimus), Alecensa (Alectinib), Alectinib, Alimta (Pemetrexed Disodium), Alunbrig (Brigatinib), Atezolizumab, Avastin (Bevacizumab), Bevacizumab, Brigatinib, Capmatinib Hydrochloride, Carboplatin, Cemiplimab-rwlc, Ceritinib, Crizotinib, Cyramza (Ramucirumab), Dabrafenib Mesylate, Dacomitinib, Docetaxel, Doxorubicin Hydrochloride, Durvalumab, Entrectinib, Erlotinib Hydrochloride, Everolimus, Gavreto (Pralsetinib), Gefitinib, Gilotrif (Afatinib Dimaleate), Gemcitabine Hydrochloride, Gemzar (Gemcitabine Hydrochloride), Imfinzi (Durvalumab), InfugemAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 (Gemcitabine Hydrochloride), Ipilimumab, Iressa (Gefitinib), Keytruda (Pembrolizumab), Libtayo (Cemiplimab-rwlc), Lorbrena (Lorlatinib), Lorlatinib, Mekinist (Trametinib Dimethyl Sulfoxide), Methotrexate Sodium, Mvasi (Bevacizumab), Necitumumab, Nivolumab, Opdivo (Nivolumab), Osimertinib Mesylate, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pembrolizumab, Pemetrexed Disodium, Portrazza (Necitumumab), Pralsetinib, Ramucirumab, Retevmo (Selpercatinib), Rozlytrek (Entrectinib), Selpercatinib, Tabrecta (Capmatinib Hydrochloride), Tafinlar (Dabrafonib Mesylate), Tagrisso (Osimertinib Mesylate), Tarceva (Erlotinib Hydrochloride), Taxotere (Docetaxel), Tecentriq (Atezolizumab), Tepmetko (Tepotinib Hydrochloride), Tepotinib Hydrochloride, Trametinib Dimethyl Sulfoxide, Trexall (Methotrexate Sodium), Vizimpro (Dacomitinib), Vinorelbine Tartrate, Xalkori (Crizotinib), Yervoy (Ipilimumab), Zirabev (Bevacizumab), Zykadia (Ceritinib), Etopophos (Etoposide Phosphate), Etoposide, Hycamtin (Topotecan Hydrochloride), or Zepzelca (Lurbinectedin), or any combination thereof.

[0165] In some cases, a subject agent, is used in a combination therapy (is co-administered) with an immunomodulatory agent. Any convenient immunomodulatory agent can be used. In some cases, the immunomodulatory agent is selected from: an anti-CTLA4 antibody; an anti-PD-l / PD-L1 agent (e.g., an anti-PD-1 antibody, a PD-l-binding reagent such as a PD-L1 or PD-L2 ectodomain, an anti-PD-Ll antibody, a PD-L1 -binding reagent such as a PD-1 ectodomain, and the like); a CD40 agonist (e.g., CD40L); a 4-1BB modulator (e.g., a 4-lBB-agonist); an anti-CD47 / SIRPA agent (e.g., an anti-CD47 antibody, a CD47-binding reagent such as a SIRPA ectodomain, an anti-SIRPA antibody, a SIRPA-binding reagent such as a CD47 ectodomain, and the like); an inhibitor of TIM3 and / or CEACAM1; an inhibitor of TIM3 and / or CEACAM1; an inhibitor of BTLA and / or CD 160; and the likeKits

[0166] Provided are kits / systems for carrying out a subject method. Such kits comprise various combinations of components useful in any of the methods described elsewhere herein.

[0167] A kit can further include one or more additional reagents, where such additional reagents can be any convenient reagent. Components of a subject kit can be in separate containers; or can be combined in a single container. In some cases one or more of a kit’s components are pharmaceutically formulated for administration to a human.

[0168] In addition to above-mentioned components, a subject kit can further include instructions for using the components of the kit to practice the subject methods (e.g., dosing instructions,Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 instructions to administer the component(s) to an individual. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In some embodiments, the instructions are present as an electronic storage data fde present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.EXEMPLARY NON-LIMITING ASPECTS OF THE DISCLOSURE

[0169] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of ordinary skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below. It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.1. A method of reducing accelerated T cell differentiation in a CD4 naive T cell (CD4 Tn cell), the method comprising:overexpressing one or more transcription factors in a CD4 Tn cell, thereby reducing accelerated T cell differentiation of the CD4 Tn cell, wherein the one or more transcription factors comprise: KLF1, KLF4, IKZF1, or any combination thereof.2. A method of inducing youthful gene expression in a CD4 naive T cell (CD4 Tn cell), the method comprising:Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 overexpressing one or more transcription factors in a CD4 Tn cell, thereby inducing youthful gene expression in the CD4 Tn cell, wherein the one or more transcription factors comprise: KLF1, KLF4, IKZF1, or any combination thereof.3. A method of reducing cellular age of a CD4 naive T cell (CD4 Tn cell), the method comprising:overexpressing one or more transcription factors in a CD4 Tn cell, thereby reducing cellular age of the CD4 Tn cell, wherein the one or more transcription factors comprise: KLF1, or KLF4, or IKZF1, or any combination thereof.4. The method of any one of 1-3, wherein said one or more transcription factors comprises: (a) IKZF1 and KLF4; or (b) IKZF1 and KLF1.5. The method of any one of 1-4, wherein said overexpressing comprises introducing one or more nucleic acids encoding the one or more transcription factors into the CD4 Tn cell.6. The method of 5, wherein said one or more nucleic acids comprise one or more mRNAs encoding the one or more transcription factors.7. The method of 6, wherein the one or more mRNAs comprise one or more base modifications, or one or more modified intemucleotide linkages, or one or more sugar modifications, or any combination thereof.8. The method of 6, wherein the one or more mRNAs comprise pseudouridine, or 5-methylcytosine (m5C), or N6-methyladenosine (m6A), or N1-methyladenosine (nfA), or any combination thereof.9. The method of any one of 6-8, wherein the one or more mRNAs were generated via in vitro transcription.10. The method of 5, wherein said one or more nucleic acids comprise one or more DNAs comprising nucleotide sequences encoding the one or more transcription factors.11. The method of 10, wherein the nucleotide sequences encoding the one or more transcription factors are operably linked to an inducible promoter.12. The method of any one of 1-4, wherein said overexpressing comprises transducing the CD4 Tn cell with a lentivirus comprising nucleic acid(s) that encodes the one or more transcription factors.13. The method of 12, wherein the lentivirus comprises one or more DNAs comprising nucleotide sequences that: (i) encode the one or more transcription factors; and (ii) are operably linked to an inducible promoter.Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 14. The method of any one of 1-4, wherein said overexpressing comprises providing a CRISPRa fusion protein and one or more guide RNAs to the CD4 Tn cell, thereby stimulating expression of the one or more transcription factors.15. The method of any one of 5-11, wherein said overexpressing comprises contacting the CD4 Tn cell with a lipid nanoparticle (LNP) comprising the one or more nucleic acids.16. The method of 15, wherein the one or more nucleic acids comprise one or more in vitro transcribed mRNAs encoding the one or more transcription factors, and wherein the one or more mRNAs comprise pseudouridine, or 5-methylcytosine (m3C), or any combination thereof.17. The method of 14, wherein said overexpressing comprises contacting the CD4 Tn cell with a lipid nanoparticle (LNP) comprising: (a) the CRISPRa fusion protein or a nucleic acid encoding the CRISPRa fusion protein; and / or (b) the one or more guide RNAs or nucleic acid(s) encoding the one or more guide RNAs.18. The method of any one of 15-17, wherein the lipid nanoparticle comprises lipids 4A3-SC8, DODAP, DOPE and cholesterol.19. The method of any one of 5-11, wherein said overexpressing comprises contacting the CD4 Tn cell with a virus or virus-like particle (VLP) comprising the one or more nucleic acids.20. The method of 19, wherein the one or more nucleic acids comprise one or moreDNAs comprising nucleotide sequences that: (i) encode the one or more transcription factors; and (ii) are operably linked to an inducible promoter.21. The method of 14, wherein said overexpressing comprises contacting the CD4 Tn cell with a virus or virus-like particle (VLP) comprising: (a) the CRISPRa fusion protein or a nucleic acid encoding the CRISPRa fusion protein; and / or (b) the one or more guide RNAs or nucleic acid(s) encoding the one or more guide RNAs.22. The method of any one of 1-21, wherein the CD4 Tn cell is ex vivo when said overexpressing is initiated.23. The method of 22, further comprising administering the CD4 Tn cell into an individual after said overexpressing is initiated.24. The method of 23, wherein the CD4 Tn cell is autologous to the individual.25. The method of 23, wherein the CD4 Tn cell is allogeneic to the individual.26. The method of any one of 23-25, wherein the individual is a mammal.27. The method of any one of 23-25, wherein the individual is a human.Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 28. The method of any one of 1-27, wherein the CD4 Tn cell is a CAR-T cell or a TCR-T cell either prior to, or after, said overexpressing.29. The method of any one of 5-13, and 15-16, and 18-20, wherein said overexpressing takes place in CD4 Tn cells in vivo and comprises administering the one or more nucleic acids to an individual.30. The method of 29, wherein the method comprises administering to the individual a lipid nanoparticle (LNP) comprising the one or more nucleic acids, or a virus or VLP comprising the one or more nucleic acids.31. The method of any one of 14, 17, and 21, wherein said overexpressing takes place in CD4 Tn cells in vivo and comprises administering to an individual: (a) the CRISPRa fusion protein or a nucleic acid encoding the CRISPRa fusion protein; and (b) the one or more guide RNAs or nucleic acid(s) encoding the one or more guide RNAs.32. The method of 31, wherein the method comprises administering to the individual a lipid nanoparticle (LNP) comprising (a) and (b), or a virus or VLP comprising (a) and (b).33. The method of any one of 23-32, wherein the individual has an infectious disease, an autoimmune disease or disorder, or a cancer.34. The method of 33, wherein the individual has rheumatoid arthritis, a solid tumor cancer, chronic lymphocytic leukemia (CLL), a viral infection, a chronic HIV-1 Infection, a bacterial infection, or any combination thereof.35. The method of any one of 23-34, wherein said administering comprises systemic or intratumoral administration.36. The method of any one of 23-34, wherein said administering comprises intravenous, intraperitoneal, subcutaneous injection, or intratumoral injection.37. A CD4 naive T cell engineered to overexpress one or more transcription factors, wherein said one or more transcription factors comprises: KLF1, KLF4, IKZF1, or any combination thereof.38. The CD4 naive T cell of 37, comprising one or more exogenous nucleic acids encoding the one or more transcription factors.39. The CD4 naive T cell of 37 or 38, wherein the CD4 Tn cell is a CAR-T cell or a TCR-T cell.Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 40. A composition, comprising one or more nucleic acids encoding one or more transcription factors, wherein the one or more transcription factors comprise: KLF1, KLF4, IKZF1, or any combination thereof.41. The composition of 40, wherein the one or more nucleic acids comprise one or more mRNAs encoding the one or more transcription factors.42. The composition of 41, wherein the one or more mRNAs comprise one or more base modifications, or one or more modified intemucleotide linkages, or one or more sugar modifications, or any combination thereof.43. The composition of 41, wherein the one or more mRNAs comprise pseudouridine, or 5-methylcytosine (m5C), or N6-methyladenosine (m6A), or N1-methyladenosine (m'A), or any combination thereof.44. The composition of any one of 41-43, wherein the one or more mRNAs are in vitro transcribed mRNAs.45. The composition of 40, wherein said one or more nucleic acids comprise one or more DNAs comprising nucleotide sequences that: (i) encode the one or more transcription factors, and (ii) are operably linked to a heterologous promoter.46. The composition of any one of 40-45, comprising a lipid nanoparticle (LNP) comprising the one or more nucleic acids.47. The composition of 40, comprising a lentiviral DNA vector comprising nucleotide sequences that: (i) encode the one or more transcription factors, and (ii) are operably linked to a heterologous promoter.48. The composition of any one of 40-45, comprising a virus or virus-like particle (VLP) comprising the one or more nucleic acids.49. The composition of any one of 40-48, further comprising a pharmaceutically acceptable excipient.50. A method of reducing accelerated T cell differentiation in a CD4 naive T cells (CD4 Tn cells) of an individual, the method comprising:overexpressing one or more transcription factors in CD4 Tn cells of an individual according to the method of any one of 1 and 4-32, thereby reducing accelerated T cell differentiation of the CD4 Tn cells.51. A method of inducing youthful gene expression in a CD4 naive T cells (CD4 Tn cells) of an individual, the method comprising:Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 overexpressing one or more transcription factors in CD4 Tn cells of an individual according to the method of any one of 2 and 4-32, thereby inducing youthful gene expression in the CD4 Tn cells.52. A method of reducing cellular age of CD4 naive T cells (CD4 Tn cells) of an individual, the method comprising:overexpressing one or more transcription factors in CD4 Tn cells of an individual according to the method of any one of 3-32, thereby reducing cellular age of the CD4 Tn cells.53. A method of treatment, the method comprising:overexpressing one or more transcription factors in CD4 naive T cells (CD4 Tn cells) according to the method of any one of 1-32, and administering the CD4 Tn cells to an individual in need thereof after said overexpressing has been initiated, thereby treating the individual. 54. The method of 53, wherein said overexpressing results in enhanced CD4 Tn cell function.55. The method of any one of 50-54, wherein the individual has an infectious disease, an autoimmune disease or disorder, or a cancer.56. The method of any one of 50-54, wherein the individual has rheumatoid arthritis, a solid tumor cancer, chronic lymphocytic leukemia (CLL), a viral infection, a chronic HIV-1 Infection, a bacterial infection, or any combination thereof.EXPERIMENTAL EXAMPLES

[0170] The following examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0171] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

[0172] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Cold Spring Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral VectorsAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits for methods referred to in, or related to, this disclosure are available from commercial vendors such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and the like, as well as repositories such as e g., Addgene, Inc., American Type Culture Collection (ATCC), and the likeExample 1Single cell pooled screens with T cells from aged donors revealed KLF1, KLF4 and IKZF1 restore a youthful gene expression profile

[0173] A single cell pooled screening methodology was used to identify transcription factors that restored youthful gene expression. CD3+ T cells were isolated from PBMCs of a panel of young and old donors. The cells were subsequently activated using TCR stimulation and transduced with a pool of lentivirus that encodes an inducible gene circuit to express each transcription factor. The TFs were then activated for 4 days followed by a 3 day chase period. At the conclusion of the experiment, T cells were sorted for expression of the TF expression machinery and subsequently profiled using single cell RNA sequencing (Figure 1).

[0174] To discover transcription factors that restored youthful gene expression, a method to predict cell age was developed by profiling a large panel of young (<30 years old) and aged (>60 years old) donors using single cell RNA sequencing. To measure cell age, a semi-supervised machine learning model was trained to predict the binary age of a T cell donor (young or old) from cell profiles. The model could robustly predict human T cell age from single cell profiles in a held-out donor task where models are trained on cells from a subset of donors, then used to predict cell age on a set of new donors. Using the cell age model, we predicted the apparent age of old cells at the conclusion of the experiment and interpreted the predictions as cell age scores (young mean of 0, old mean of 1). If a TF set significantly reduced cell age scores, we interpreted this result as a restoration of youthful gene expression.

[0175] Transient expression of KLEI, KLF4 an I KZ Fl and combinations of IKZF1 AND KLF4, IKZF1 AND KLF1 significantly reduced the apparent age of T cells from aged donors (Figure 2).Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 Example 2CD4 Tn cells from aged donors exhibit accelerated differentiation

[0176] CD4 naive T cells (CD4 Tn) were isolated from a panel of young and aged PBMCs donors using fluorescence assisted cell sorting. The cells were activated using a plate bound anti-CD3 antibody as well as a soluble co-stimulatory antibody against CD28. The cells were then differentiated for 10 days with regular media changes. Differentiation state was then quantified at the end of the experiment using cell surface markers.

[0177] CD4 T cells from aged donors exhibited a marked downregulation of CD45RA protein expression after 10 days in culture while young donors exhibited a significantly higher expression level of CD45RA (Figure 3).Example 3Transient expression of rejuvenating transcription factors (KLF1, KLF4, IKZF1)

[0178] Using a similar assay as above, we activated CD4 naive T cells and the following day the cells were transduced with lentiviral constructs to enable inducible expression of KLF1, KLF4, IKZF1 mKate2 and other TFs in an arrayed format. Following the same timeline as the pooled screen, the TFs were pulsed for 4 days and then T cell differentiation state was measured 4 days later (Day 10 of the experiment). KLF1, KLF4 and IKZF1 independently normalized the T cell differentiation state to a youthful phenotype across a panel of 3 aged donors (Figure 4).

[0179] Accelerated T cell differentiation has been implicated in the progression of rheumatoid arthritis [ref. 1-3] and the presence of less differentiated T cells subsets improves outcomes in both infectious disease and oncology [ref. 4-6],

[0180] References(1) Weyand et al., Nat Rev Rheumatol. 2017 May;13(5):291-301(2) Weyand et al., Free Radic Biol Med. 2018 Sep;125:36-43(3) Wu et al., Immunometabolism. 2020;2(2):e200017(4) Alizadeh et al., Cancer Immunol Res. 2019 May;7(5):759-772(5) Fraietta et al., Nat Med. 2018 May;24(5):563-571(6) Ribeiro et al., J Virol. 2014 Dec;88(23): 13836-44Atty Docket No.: NEWL-003 / 01WO 42005 / 9 Methods

[0181] The following methods were used in Examples 1-3 above.T cell culture

[0182] Human primary peripheral blood mononuclear cells (PMBCs) were purchased from commercial sources and isolated from leukopaks using Ficoll-Plaque PLUS (Fisher Scientific). Isolated PBMCs were cryopreserved and stored in liquid nitrogen. For each experiment, PBMCs were thawed and incubated overnight in human T cell culture media (hTCM) consisting of X-Vivo-15 medium (Lonza) supplemented with 5% fetal bovine serum (FBS), 50 pM 2-mercaptoethanol, and 10 mM N-acetyl-l-cysteine, as previously described. T cells were isolated from PBMCs using EasySep CD3 isolation kits (StemCell Technologies) and cultured in hTCM containing 500 lU / mL IL-2 (R&D Systems). For T cell transduction, we developed a lentiviral construct from an existing 3rd generation lentiviral vector (VectorBuilder). We inserted promoters and selectable markers by Gibson assembly to create a parent vector, then inserted ORFs and barcodes using sequential Gibson cloning steps (PRX vector). Lentivirus was packaged using the Lipofectamine 3000 system (ThermoFisher) and 2nd generation packaging plasmids (Cellecta) in HEK293T cells as previously described. Particles were concentrated 100-fold using Lenti-X concentrater (Takara) before delivery to T cells.Pooled screening

[0183] Freshly isolated T cells were activated with anti-CD3 / anti-CD28 Dynabeads (ThermoFisher) for 24 hours. After 24 hours, lentivirus expressing both TFs and the Tet activator were added to the culture media that contained 500 lU / mL IL -2 and 1 ug / mL doxycycline. On the following day, the Dynabeads were removed and lentivirus media was replaced with fresh media containing IL-2 and doxycycline. After 48 hours, the media was refreshed with media containing IL-2 and doxycycline. One day later, doxycycline containing media was removed and cell samples were collected for single cell profiling. 48 hours later the media exchanged with fresh hTCM. 24 hours later cell samples were collected for single cell profiling.Single cell RNA-sequencing

[0184] At each time point, we sorted cells expressing both our perturbation vectors (eGFP+) and tetracycline transactivator machinery (BFP+ or tNGFR+). We profiled single cells using the lOxAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 Genomics NextGem single cell gene expression 3' RNA-seq chemistry (HT v3.1) on the Chromium X system (lOx Genomics). We pooled multiple donors and loaded them into common reactions to allow for genotype-based filtering of multiplets in our final analysis, as previously described (Kang et al. Nat Biotechnol 36, 89-94 (2018)). We enriched for perturbation barcodes using PCR as previously described [Hill et al., Nat Methods, Volume 15, Issue 4, April 2018, Pages 271-274], We performed quality control using a Bioanalyzer with High Sensitivity DNA kits (Agilent) and DNA sequencing using Illumina Nextseq and Novaseq systems as described in the lOx Genomics protocol (Illumina), targeting 20,000 reads per cell for gene expression libraries and 5,000 reads per cell for perturbation barcodes.Genomics preprocessing

[0185] Following sequencing, reads were aligned to a reference transcriptome containing both mouse and human transcriptomes. Cells harboring a high number of mouse transcripts were filtered from all subsequent analyses. Human cells were demultiplexed and assigned to specific donors using standard software that extracts cell genotypes from sequencing reads [Huang et al., Bioinformatics, Volume 37, Issue 23, December 2021, Pages 4569-4571; Huang et al., Genome Biol 20, 273 (2019)]. Based on the donor assignments, we determined the original age of each cell. We detected transgene barcodes from the lentiviral vectors and determined the presence or absence of each transgene in each cell using a Gaussian mixture modeling approach from the literature [Bernstein et al., Cell Syst. 2020 Jul 22; 1 l(l):95-101.e5.].Measuring an aging gene expression program

[0186] A matrix of cells by gene counts was generated where each value in the matrix represented the number of unique molecular identifiers captured in a given cell that map to a specific gene. A logistic regression model was implemented to predict a binary age label from the vector of gene counts for each cell. The model was trained to predict the ground truth age of cells treated with control transgenes (mKate2) after binarizing ages to young (0; <35 years of age) and old (1; >60 years of age). Following training, the model was used to predict the age of all cells in the dataset and the raw logits were stored. The mean predicted age for all old cells that received a given transgene was computed.

[0187] Perturbations were then ranked by the mean predicted age, with lower predicted ages suggesting the restoration of a youthful gene expression program. Significance testing wasAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 performed by comparing the predicted ages for cells that received a given transgene to the negative control with a 2-sample t-test with unequal variance.Measurement of differentiation phenotype across a panel of young and aged donors

[0188] CD4 naive T (Tn) cells were isolated from PBMCs with a two-step procedure. Immediately after thawing PBMCs, CD14+ cells were depleted using MACS isolation (Miltenyi 130-050-201). The negative fraction was incubated overnight in a 37°C incubator at 5% CO2. CD4 Tn cells were subsequently incubated with antibodies (CD3-FITC, Biolegend 317306; CD4-BV605, Biolegend 317438; CD8a-BV711, Biolegend 344734; CD27-APC, Biolegend 302810; CD45RA-PE / Cy7, Biolegend 304126; CD95-BV421, Biolegend 305624; LIVE / DEAD NearIR, ThermoFisher L34994) and sorted using FACS to isolate a CD3+ / CD4+ / CD45RA+ / CD27+ / CD95- population. CD4 Tn cells were activated using plate bound 10 ug / mL OKT3 (Biolegend 317347) and 2 ug / mL soluble CD28 (Biolegend 102121). The hTCM was exchanged every other day for a period of 10 days prior to quantification of T cell differentiation state. T cell differentiation was quantified using CD4-BV605 (Biolegend 317438), CD45RA-BV711 (BioLegend 304138), CD27-APC (Biolegend 302810) and LIVE / DEAD NearIR (ThermoFisher L34994). Tn-like cells were defined as CD4+ / CD27+ / CD45RA+ and Tcm cells were defined as CD4+ / CD27+ / CD45RA-.Validation of CD4 naive T cell differentiation assay

[0189] CD4 naive T cells were isolated and activated the same as above (i.e. Measurement of differentiation phenotype across a panel of young and aged donors). After 24 hours of activation, the cells were transduced with lentivirus that expressed a PRX vector for a specific TF and lentivirus that expressed Tet transactivator. After a 24 hour incubation with virus, the virus was removed and the media was refreshed with hTCM containing 50 lU / mL IL-2 and 1 ug / mL doxycyline. The media was refreshed with hTCM containing 50 lU / mL IL-2 and 1 ug / mL doxycycline for four days. After day 6, the media was refreshed every other day with hTCM contain 50 lU / mL IL-2 for five days. We quantified %Tn-like and %Tcm for each TF across 3 old donors. To identify TFs that were significantly different from our negative control perturbation, we used a linear mixed effects model of the form: % Tn ~ TF + (1 / Donor), whereAtty Docket No.: NEWL-003 / 01WQ 42005 / 9 TF is a fixed effect for the TF payload treatment, (1 / Donor) is a random effect for the cell donor, and % Tn is the percentage of Tn-like cells in the sample.

[0190] Significance testing on the fixed effect was performed using the Wald test. Multiple hypothesis testing was corrected for using the Benj amini -Hochberg procedure to generate q values from p values and consider effects where q < 0.1 to be significant.

[0191] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0192] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0193] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

Atty Docket No.: NEWL-003 / 01WQ 42005 / 9CLAIMSWhat is claimed is:

1. A method of reducing accelerated T cell differentiation in a CD4 naive T cell (CD4 Tn cell), the method comprising:overexpressing one or more transcription factors in a CD4 Tn cell, thereby reducing accelerated T cell differentiation of the CD4 Tn cell, wherein the one or more transcription factors comprise: KLF1, KLF4, IKZF1, or any combination thereof.

2. A method of inducing youthful gene expression in a CD4 naive T cell (CD4 Tn cell), the method comprising:overexpressing one or more transcription factors in a CD4 Tn cell, thereby inducing youthful gene expression in the CD4 Tn cell, wherein the one or more transcription factors comprise: KLF1, KLF4, IKZF1, or any combination thereof.

3. A method of reducing cellular age of a CD4 naive T cell (CD4 Tn cell), the method comprising:overexpressing one or more transcription factors in a CD4 Tn cell, thereby reducing cellular age of the CD4 Tn cell, wherein the one or more transcription factors comprise: KLF1, or KLF4, or IKZF1, or any combination thereof.

4. The method of any one of claims 1-3, wherein said one or more transcription factors comprises: (a) IKZF1 and KLF4; or (b) IKZF1 and KLF1.

5. The method of any one of claims 1-4, wherein said overexpressing comprises introducing one or more nucleic acids encoding the one or more transcription factors into the CD4 Tn cell.

6. The method of claim 5, wherein said one or more nucleic acids comprise one or more mRNAs encoding the one or more transcription factors.Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 7. The method of claim 6, wherein the one or more mRNAs comprise one or more base modifications, or one or more modified internucleotide linkages, or one or more sugar modifications, or any combination thereof.

8. The method of claim 6, wherein the one or more mRNAs comprise pseudouridine, or 5-methylcytosine (m5C), or N6-methyl adenosine (m6A), or Nkmethyladenosine (mxA), or any combination thereof.

9. The method of any one of claims 6-8, wherein the one or more mRNAs were generated via in vitro transcription.

10. The method of claim 5, wherein said one or more nucleic acids comprise one or more DNAs comprising nucleotide sequences encoding the one or more transcription factors.

11. The method of claim 10, wherein the nucleotide sequences encoding the one or more transcription factors are operably linked to an inducible promoter.

12. The method of any one of claims 1-4, wherein said overexpressing comprises transducing the CD4 Tn cell with a lentivirus comprising nucleic acid(s) that encodes the one or more transcription factors.

13. The method of claim 12, wherein the lentivirus comprises one or more DNAs comprising nucleotide sequences that: (i) encode the one or more transcription factors; and (ii) are operably linked to an inducible promoter.

14. The method of any one of claims 1-4, wherein said overexpressing comprises providing a CRISPRa fusion protein and one or more guide RNAs to the CD4 Tn cell, thereby stimulating expression of the one or more transcription factors.

15. The method of any one of claims 5-11, wherein said overexpressing comprises contacting the CD4 Tn cell with a lipid nanoparticle (LNP) comprising the one or more nucleic acids.Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 16. The method of claim 15, wherein the one or more nucleic acids comprise one or more in vitro transcribed mRNAs encoding the one or more transcription factors, and wherein the one or more mRNAs comprise pseudouridine, or 5-methylcytosine (nEC), or any combination thereof.

17. The method of claim 14, wherein said overexpressing comprises contacting the CD4 Tn cell with a lipid nanoparticle (LNP) comprising: (a) the CRISPRa fusion protein or a nucleic acid encoding the CRISPRa fusion protein; and / or (b) the one or more guide RNAs or nucleic acid(s) encoding the one or more guide RNAs.

18. The method of any one of claims 15-17, wherein the lipid nanoparticle comprises lipids 4A3-SC8, DODAP, DOPE and cholesterol.

19. The method of any one of claims 5-11, wherein said overexpressing comprises contacting the CD4 Tn cell with a virus or virus-like particle (VLP) comprising the one or more nucleic acids.

20. The method of claim 19, wherein the one or more nucleic acids comprise one or more DNAs comprising nucleotide sequences that: (i) encode the one or more transcription factors; and (ii) are operably linked to an inducible promoter.

21. The method of claim 14, wherein said overexpressing comprises contacting the CD4 Tn cell with a virus or virus-like particle (VLP) comprising: (a) the CRISPRa fusion protein or a nucleic acid encoding the CRISPRa fusion protein; and / or (b) the one or more guide RNAs or nucleic acid(s) encoding the one or more guide RNAs.

22. The method of any one of claims 1-21, wherein the CD4 Tn cell is ex vivo when said overexpressing is initiated.

23. The method of claim 22, further comprising administering the CD4 Tn cell into an individual after said overexpressing is initiated.

24. The method of 23, wherein the CD4 Tn cell is autologous to the individual.Atty Docket No.: NEWL-003 / 01WQ 42005 / 925. The method of 23, wherein the CD4 Tn cell is allogeneic to the individual.

26. The method of any one of 23-25, wherein the individual is a mammal.

27. The method of any one of 23-25, wherein the individual is a human.

28. The method of any one of claims 1-27, wherein the CD4 Tn cell is a CAR-T cell or a TCR-T cell either prior to, or after, said overexpressing.

29. The method of any one of claims 5-13, and 15-16, and 18-20, wherein said overexpressing takes place in CD4 Tn cells in vivo and comprises administering the one or more nucleic acids to an individual.

30. The method of claim 29, wherein the method comprises administering to the individual a lipid nanoparticle (LNP) comprising the one or more nucleic acids, or a virus or VLP comprising the one or more nucleic acids.

31. The method of any one of claims 14, 17, and 21, wherein said overexpressing takes place in CD4 Tn cells in vivo and comprises administering to an individual: (a) the CRISPRa fusion protein or a nucleic acid encoding the CRISPRa fusion protein; and (b) the one or more guide RNAs or nucleic acid(s) encoding the one or more guide RNAs.

32. The method of claim 31, wherein the method comprises administering to the individual a lipid nanoparticle (LNP) comprising (a) and (b), or a virus or VLP comprising (a) and (b).

33. The method of any one of claims 23-32, wherein the individual has an infectious disease, an autoimmune disease or disorder, or a cancer.

34. The method of claim 33, wherein the individual has rheumatoid arthritis, a solid tumor cancer, chronic lymphocytic leukemia (CLL), a viral infection, a chronic HIV-1 Infection, a bacterial infection, or any combination thereof.

35. The method of any one of claims 23-34, wherein said administering comprises systemic or intratumoral administration.Atty Docket No.: NEWL-003 / 01WQ 42005 / 936. The method of any one of claims 23-34, wherein said administering comprises intravenous, intraperitoneal, subcutaneous injection, or intratumoral injection.

37. A CD4 naive T cell engineered to overexpress one or more transcription factors, wherein said one or more transcription factors comprises: KLF1, KLF4, IKZF1, or any combination thereof.

38. The CD4 naive T cell of claim 37, comprising one or more exogenous nucleic acids encoding the one or more transcription factors.

39. The CD4 naive T cell of claim 37 or claim 38, wherein the CD4 Tn cell is a CAR-T cell or a TCR-T cell.

40. A composition, comprising one or more nucleic acids encoding one or more transcription factors, wherein the one or more transcription factors comprise: KLF1, KLF4, IKZF1, or any combination thereof.

41. The composition of claim 40, wherein the one or more nucleic acids comprise one or more mRNAs encoding the one or more transcription factors.

42. The composition of claim 41, wherein the one or more mRNAs comprise one or more base modifications, or one or more modified intemucleotide linkages, or one or more sugar modifications, or any combination thereof.

43. The composition of claim 41, wherein the one or more mRNAs comprise pseudouridine, or 5-methylcytosine (m5C), or N6-methyladenosine (m6A), or N^methyladenosine (in1A), or any combination thereof.

44. The composition of any one of claims 41-43, wherein the one or more mRNAs are in vitro transcribed mRNAs.Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 45. The composition of claim 40, wherein said one or more nucleic acids comprise one or more DNAs comprising nucleotide sequences that: (i) encode the one or more transcription factors, and (ii) are operably linked to a heterologous promoter.

46. The composition of any one of claims 40-45, comprising a lipid nanoparticle (LNP) comprising the one or more nucleic acids.

47. The composition of claim 40, comprising a lentiviral DNA vector comprising nucleotide sequences that: (i) encode the one or more transcription factors, and (ii) are operably linked to a heterologous promoter.

48. The composition of any one of claims 40-45, comprising a virus or virus-like particle (VLP) comprising the one or more nucleic acids.

49. The composition of any one of claims 40-48, further comprising a pharmaceutically acceptable excipient.

50. A method of reducing accelerated T cell differentiation in a CD4 naive T cells (CD4 Tn cells) of an individual, the method comprising:overexpressing one or more transcription factors in CD4 Tn cells of an individual according to the method of any one of claims 1 and 4-32, thereby reducing accelerated T cell differentiation of the CD4 Tn cells.

51. A method of inducing youthful gene expression in a CD4 naive T cells (CD4 Tn cells) of an individual, the method comprising:overexpressing one or more transcription factors in CD4 Tn cells of an individual according to the method of any one of claims 2 and 4-32, thereby inducing youthful gene expression in the CD4 Tn cells.

52. A method of reducing cellular age of CD4 naive T cells (CD4 Tn cells) of an individual, the method comprising:Atty Docket No.: NEWL-003 / 01WQ 42005 / 9 overexpressing one or more transcription factors in CD4 Tn cells of an individual according to the method of any one of claims 3-32, thereby reducing cellular age of the CD4 Tn cells.

53. A method of treatment, the method comprising:overexpressing one or more transcription factors in CD4 naive T cells (CD4 Tn cells) according to the method of any one of claims 1-32, and administering the CD4 Tn cells to an individual in need thereof after said overexpressing has been initiated, thereby treating the individual.

54. The method of claim 53, wherein said overexpressing results in enhanced CD4 Tn cell function.

55. The method of any one of claims 50-54, wherein the individual has an infectious disease, an autoimmune disease or disorder, or a cancer.

56. The method of any one of claims 50-54, wherein the individual has rheumatoid arthritis, a solid tumor cancer, chronic lymphocytic leukemia (CLL), a viral infection, a chronic HIV-1 Infection, a bacterial infection, or any combination thereof.

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