Cell delivery compositions and methods of use

Abstract

The present disclosure provides a cargo delivery fusion polypeptide comprising an endosomolytic polypeptide and a cell penetrating polypeptide, and a composition comprising said cargo delivery fusion polypeptide. The present disclosure also provides a method of delivering a cargo to a target eukaryotic cell using a composition comprising an amphipathic polypeptide.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 282,622, filed November 23, 2021, the entire contents of which are incorporated herein by reference.

[0002] Statement regarding federally funded research This invention was made with Government support under AI150552 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0003] INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY The Sequence Listing is provided herewith as Sequence Listing XML "BERK-452WO_SEQ_LIST", created on November 21, 2022 and 230KB in size. The Sequence Listing XML is hereby incorporated by reference in its entirety. [Background technology]

[0004] Introduction Biopolymers have great potential as therapeutic agents. For example, DNA vaccines have been extensively studied as they provide a rapid and inexpensive approach to vaccination against a wide range of viral pathogens. In DNA vaccines, DNA encoding viral proteins is delivered to the nucleus of cells, where it is transcribed to produce viral proteins that are recognized by the immune system. The efficacy of DNA vaccines is weakened by poor delivery of DNA to target cells, which does not elicit a sufficiently effective immune response to provide effective vaccination. Therefore, DNA vaccines have been administered by in vivo electroporation, a painful method that requires specialized equipment and may require repeated administrations.

[0005] Genome editing holds great therapeutic promise in correcting genetic mutations underlying disease and in preventing or treating non-genetic diseases. Delivery of genome editing enzymes, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (CRISPR-Cas) effector polypeptides, into the cytoplasm or nucleus of cells that need to be manipulated remains the biggest hurdle. Delivery of CRISPR-Cas effector polypeptides in the form of ribonucleoprotein (RNP) complexes containing guide RNA offers many advantages compared to other approaches, such as the use of viral vectors carrying DNA or lipid nanoparticles carrying mRNA.

[0006] There is a need in the art for compositions and methods for delivering cargoes, such as DNA vaccines and genome editing enzymes, into eukaryotic cells. Summary of the Invention

[0007] overview The present invention provides a cargo delivery fusion polypeptide comprising an endosomolytic polypeptide and a cell penetrating polypeptide, and a composition comprising said cargo delivery fusion polypeptide. The present invention also provides a method of delivering a cargo to a target eukaryotic cell using a composition comprising an amphipathic polypeptide. [Brief description of the drawings]

[0008] [Figure 1-1] Various cargo delivery fusion polypeptides (SEQ ID NOs: 1-37 and 40-60, respectively) are shown. [Figure 1-2] See description of Figure 1-1. [Diagram 2] FIG. 1 is a schematic diagram of CRISPR-Cas9 delivery to target cells using a cargo delivery fusion polypeptide of the invention. [Diagram 3]Screening of peptides 1-37 (shown in FIG. 1) for Cas9-mediated knockout (KO) at the β-2 microglobulin (β2M) locus in human primary CD4+ T cells as measured by flow cytometry. [Figure 4] Screening of peptides for Cas9-mediated KO at the CD4 locus in human primary CD4+ T cells assessed by flow cytometry (Figure 4A) or deep sequencing (Figure 4B). [Diagram 5] Screening of peptide numbers 40-60 (shown in Figure 1) for Cas9-mediated KO at the β2M locus in human primary CD4+ T cells as measured by flow cytometry (Figure 5A), and viable cell counts from each treatment condition as measured by live / dead fixable violet staining and flow cytometry (Figure 5B). [Figure 6] Shown are the results of screening peptides no. 1–37 (depicted in Figure 1) that enable Cas9-mediated non-homologous end joining (NHEJ) at the erythroid-specific BCL11a enhancer locus in CD34+ hematopoietic stem and progenitor cells (HSPCs), as measured by deep sequencing at peptide concentrations of either 5 µM or 10 µM. [Figure 7] Screening of peptides no. 40–58 (shown in Figure 1) for promoting Cas9-3x-NLS-mediated NHEJ in HSPCs as measured by deep sequencing at a final peptide concentration of either 5 µM or 10 µM (Figure 7A), HSPC viability under each condition (Figure 7B), and the total score of each peptide calculated by [(% viability × % NHEJ) × 100] (Figure 7C) are shown. [Figure 8] Figures 8A-C show the effect of non-ionic surfactant additives on peptide-Cas9 formulations. [Figure 9] Screening of Cas9-mediated genome editing in primary mouse neural progenitor cells (NPCs) from Ai9 mice as measured by tdTomato signal detected by flow cytometry. [Figure 10]Screening of Cas9-mediated genome editing in primary mouse neural progenitor cells (NPCs) from Ai9 mice as measured by tdTomato signal by flow cytometry. [Figure 11] 1 shows editing of the CD45 locus in B cells, T cells, and NK cells by peptide co-culture or electroporation. [Figure 12] We show the levels of β2M KO in T and B cells cultured separately or as co-cultures of T and B cells as measured by flow cytometry (Figure 12A), as well as the editing ratio of T cells to B cells in several treatment conditions (Figure 12B). [Figure 13] Shown are the percentage of primary human B cells exhibiting β2M KO under different treatment conditions (Figure 13A), representative flow scatter plots showing the gating strategy for analyzing β2M KO under the four conditions (Figure 13B), and viable cell counts of cells 3 days after each treatment measured by flow cytometry (Figure 13C). [Figure 14] 14A-C show knock-in (KI) of a FLAG tag into the CD5 locus in primary human CD4+ T cells by homologous directed repair (HDR). [Figure 15] Figure 15A-C shows knock-in of 1928z chimeric antigen receptor (CAR) at the T cell receptor (TCR) locus of CD4+ T cells. TRAC ribonucleoprotein (RNP) was delivered by co-incubation with A5K peptide (peptide number 22) to KO TRAC, and the CAR locus was delivered by AAV6 at four different time points (30 min before, simultaneously, 30 min after, or 2 h after RNP treatment). [Figure 16] 16A-C show sequential editing of CD4+ primary T cells at three genomic loci (TRAC, CD5, β2M) by co-incubation of Cas9 RNP with 10 μM, 15 μM, or 20 μM of A5K peptide (peptide number 22). [Figure 17] 17A-B show peptide-mediated editing in T cells under various stimulation conditions. [Figure 18]Figures 18A-D show knock-in of 1928z-CAR at the TCR locus in CD3+ bulk T cells followed by sequential KO. [Figure 19] Figures 19A-D show knock-in of 1928z-CAR at the TCR locus in CD3+ bulk T cells or cells treated for KO only (no AAV-mediated KI) followed by sequential KO. [Figure 20] 1 shows adenine to guanine base editing at the CCR5 locus in primary human T cells delivered by peptide co-incubation with A5K peptide (peptide number 22). [Figure 21] 1 shows adenine to guanine base editing at the CCR5 locus in primary human T cells delivered by peptide co-incubation with A5K peptide (peptide number 22). [Figure 22] 13 shows base editing in primary human HSPCs at the erythroid-specific BCL11a enhancer locus. [Figure 23] FIG. 1 is a schematic diagram of peptide-mediated DNA delivery in the context of DNA vaccines. [Figure 24] 1 shows peptide-mediated DNA delivery and protein expression in DC2.4 cells. [Diagram 25] Figure 1 shows antibody titers measured by enzyme-linked immunosorbent assay (ELISA) in mice injected with plasmid DNA encoding the receptor binding domain (RBD) of the spike protein from SARS CoV-2 with or without the addition of 200 pmol of E5-TAT peptide. [Figure 26] The nucleotide sequences of the gRNAs used in each Example are shown (SEQ ID NOs: 125 to 136, respectively). [Figure 27A] 1 shows the amino acid sequence of a fusion polypeptide comprising a Cas9 polypeptide and a base editor. [Figure 27B] 1 shows the amino acid sequence of a fusion polypeptide comprising a Cas9 polypeptide and a base editor. [Figure 27C]1 shows the amino acid sequence of a fusion polypeptide comprising a Cas9 polypeptide and a base editor. [Figure 28A] 1 shows the amino acid sequence of the Streptococcus pyogenes Cas9 polypeptide (SEQ ID NO: 143). [Figure 28B] 1 shows the amino acid sequence of the Staphylococcus aureus Cas9 polypeptide (SEQ ID NO: 144). [Figure 28C] 1 shows the amino acid sequence of Cas12a polypeptide (SEQ ID NO: 145). [Figure 28D] 1 shows the amino acid sequence of Cas12a polypeptide (SEQ ID NO: 146). [Figure 29A] 1 shows the amino acid sequence (SEQ ID NO: 147) of the fusion protein used in some of the Examples. [Figure 29B] 1 shows the amino acid sequence (SEQ ID NO: 148) of the fusion protein used in some of the Examples. [Figure 29C] 1 shows the amino acid sequence (SEQ ID NO: 149) of the fusion protein used in some of the Examples. [Figure 29D] The amino acid sequence of the fusion protein used in some of the examples (SEQ ID NO: 150) is shown. [Figure 29E] The amino acid sequence (SEQ ID NO: 151) of the fusion protein used in some of the Examples is shown. [Figure 30-1] Various cargo-delivery fusion polypeptides are shown. [Figure 30-2] See description of Figure 30-1. [Diagram 31] 1 shows the amino acid sequence (SEQ ID NO: 152) of the base editor ("ABE8e-SpCas9-NGG dTadA / TadA8e dimer with C-terminal BP-SV40 / Nuc"). [Diagram 32] The spacer sequences of the guide RNAs used in each Example are shown (SEQ ID NOs: 153 to 156, respectively). [Figure 33A] 1 shows genome editing upon delivery of RNP to T cells using various cargo delivery fusion polypeptides. [Figure 33B] FIG. 1 shows cell viability when RNP is delivered to T cells using various cargo delivery fusion polypeptides. [Figure 34A] 1 shows genome editing upon delivery of RNP to T cells using various cargo delivery fusion polypeptides. [Figure 34B] FIG. 1 shows cell viability when RNP is delivered to T cells using various cargo delivery fusion polypeptides. [Diagram 35] Figure 1 shows genome editing and cell viability when RNPs are delivered into primary T cells using various cargo-delivery fusion polypeptides with different functional groups. [Figure 36-1] Genome editing and cell viability upon delivery of RNPs into human primary CD34+ hematopoietic stem and progenitor cells (HSPCs) using various cargo delivery fusion polypeptides. [Figure 36-2] See description of Figure 36-1. [Figure 37] Genome editing and cell viability are shown when RNPs are delivered to HSPCs using different cargo delivery fusion polypeptides. [Figure 38] Genome editing and cell viability are shown when RNPs are delivered to neural progenitor cells (NPCs) using various cargo-delivering fusion polypeptides. [Figure 39] Figures 39A-D show peptide-mediated delivery of Cas9 RNP (i.e., delivery using a cargo-delivery fusion polypeptide) to generate tumor-killing CAR-T cells in vivo. [Figure 40A] 1 shows peptide-mediated delivery to generate tumor-killing CAR-T cells ex vivo. [Figure 40B] 1 shows peptide-mediated delivery to generate tumor-killing CAR-T cells ex vivo. [Figure 40C] 1 shows peptide-mediated delivery to generate tumor-killing CAR-T cells ex vivo. [Figure 40D] 1 shows peptide-mediated delivery to generate tumor-killing CAR-T cells ex vivo. [Diagram 41] FIG. 1 shows a comparison of editing efficiency and viability following peptide-mediated delivery or electroporation (e-por) with peptide 22 (A5K (Peptide No. 22), "PERC") of various S. pyogenes Cas9 protein constructs. [Figure 42A]

[0023] Figure 1 shows data demonstrating that peptide-mediated delivery ("PERC") supports cell viability and phenotypic maintenance during serial editing. [Figure 42B]

[0023] Figure 1 shows data demonstrating that peptide-mediated delivery ("PERC") supports cell viability and phenotypic maintenance during serial editing. [Figure 42C]

[0023] Figure 1 shows data demonstrating that peptide-mediated delivery ("PERC") supports cell viability and phenotypic maintenance during serial editing. [Fig.42D]

[0023] Figure 1 shows data demonstrating that peptide-mediated delivery ("PERC") supports cell viability and phenotypic maintenance during serial editing. [Figure 43A] 13 shows data demonstrating that PERC supports cell viability in generating modified T cells with multiple knock-in edits at different genomic loci. [Figure 43B] 13 shows data demonstrating that PERC supports cell viability in generating modified T cells with multiple knock-in edits at different genomic loci. [Figure 43C] 13 shows data demonstrating that PERC supports cell viability in generating modified T cells with multiple knock-in edits at different genomic loci. [Fig. 43D] 13 shows data demonstrating that PERC supports cell viability in generating modified T cells with multiple knock-in edits at different genomic loci. [Figure 44A] We present data demonstrating that PERC of CRISPR RNPs supports T cell engineering while minimizing induction of T cell phenotypic perturbation, particularly when compared to electroporation of RNPs. [Figure 44B]We present data demonstrating that PERC of CRISPR RNPs supports T cell engineering while minimizing induction of T cell phenotypic perturbation, particularly when compared to electroporation of RNPs. [Figure 44C] We present data demonstrating that PERC of CRISPR RNPs supports T cell engineering while minimizing induction of T cell phenotypic perturbation, particularly when compared to electroporation of RNPs. [Fig.44D] We present data demonstrating that PERC of CRISPR RNPs supports T cell engineering while minimizing induction of T cell phenotypic perturbation, particularly when compared to electroporation of RNPs. [Figure 44E] We present data demonstrating that PERC of CRISPR RNPs supports T cell engineering while minimizing induction of T cell phenotypic perturbation, particularly when compared to electroporation of RNPs. [Fig.44F] We present data demonstrating that PERC of CRISPR RNPs supports T cell engineering while minimizing induction of T cell phenotypic perturbation, particularly when compared to electroporation of RNPs. [Diagram 45] We present data demonstrating that PERC of CRISPR RNPs supports multiplex genome editing while minimizing the induction of chromosomal translocations, especially when compared with co-electroporation of RNP nucleases targeting multiple genomic loci. [Figure 46] Figures 46A-C show data demonstrating that PERC of CRISPR RNPs supports improved cell yield and stable cell growth over time following T cell engineering, especially when compared to electroporation of RNPs. [Figure 47] We present data demonstrating that CRISPR RNP PERC supports precise knock-in of genes into primary human B cells when AAV6 is used to provide the necessary DNA donor template to enable homology-directed repair (HDR). [Figure 48]We present data demonstrating that the CRISPR RNP PERC aids in precise gene knock-in into primary human NK cells when AAV6 is used to provide the necessary DNA donor template to enable HDR. [Figure 49]

[0023] Figure 1 shows data demonstrating that the CRISPR RNP PERC supports highly efficient genome editing of primary human CD34+ HSPCs at the BCL11a locus. [Figure 50] We present data demonstrating that the CRISPR RNP PERC supports highly efficient genome editing of primary human CD34+ HSPCs at the beta2 microglobulin (B2M, β2M) locus. [Figure 51] 13 shows base editing in primary human HSPCs at the erythroid-specific BCL11a enhancer locus. [Figure 52] Shows genome editing in striatal neurons in the mouse brain. [Figure 53] Figures 53A-B show quantification of neuronal editing in Ai9 mice. [Figure 54]

[0023] Figure 1 shows data demonstrating that intravenously administered formulations containing Cas9 RNP and peptides can promote genome editing of human primary T cells in vivo. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] definition The terms "polypeptide," "peptide," and "protein," as used interchangeably herein, refer to polymeric forms of amino acids of any length and can include genetically and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The terms include fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, with or without an N-terminal methionine residue, fusion proteins with heterologous and homologous leader sequences, immunologically tagged proteins, and the like.

[0010] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers that contain purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0011] "Operably linked" refers to the juxtaposition of two or more elements, in a relationship permitting the elements described by this term to function in their intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

[0012] A "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus, cosmid, or the like, to which another DNA segment, or "insert," may be added so as to bring about the replication of the added segment within a cell.

[0013] As used herein, "heterologous" refers to a nucleotide sequence or polypeptide sequence that is not found in the native (eg, naturally occurring) nucleic acid or protein, respectively.

[0014] The term "antibody" includes antibodies of any isotype, and fragments of antibodies that retain specific binding to an antigen, including, but not limited to, Fab, Fv, single chain Fv (scFv), and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies (scAbs), single domain antibodies (dAbs), single domain heavy chain antibodies, single domain light chain antibodies, nanobodies, bispecific antibodies, multispecific antibodies, and fusion proteins comprising the antigen-binding (also referred to herein as antigen-binding) portion of an antibody and a non-antibody protein.

[0015] As used herein, the terms "treatment," "treating," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic, in terms of partially or completely curing a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, including (a) preventing the onset of the disease in a subject susceptible to, but not yet diagnosed as having, the disease, (b) inhibiting the disease, i.e., preventing its development, and (c) relieving the disease, i.e., causing regression of the disease.

[0016] The terms "individual," "subject," "host," and "patient," as used interchangeably herein, refer to mammals, including but not limited to mice (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats), lagomorphs, and the like. In some cases, the individual is a human. In some cases, the individual is a non-human primate. In some cases, the individual is a rodent, such as a rat or mouse. In some cases, the individual is a lagomorph, such as a rabbit.

[0017] Before the present invention is further described, it is to be understood that the invention is not limited to the particular embodiments described, and as such may vary widely. 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 is limited only by the appended claims.

[0018] When a range of values ​​is provided, unless the context clearly dictates otherwise, it is to be understood that each intermediate value between the upper and lower limit of that range, and any other stated or intermediate value within that stated range, to the tenth of the unit of the lower limit, 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.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials for which the publications are cited.

[0020] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "an amphiphilic polypeptide" includes a plurality of such polypeptides, reference to "the cell" refers to one or more capsules and their equivalents known to those skilled in the art, and so forth. It should be further noted that the claims may be drafted to exclude any element. In such cases, this statement is intended to serve as a predicate to the recitation of elements in the claims or the use of exclusive language such as "solely," "only," and the like in connection with the use of a "negative" limitation.

[0021] Certain features of the invention that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described in the context of a single embodiment for brevity may also be provided separately or in any suitable subcombination. All combinations of the embodiments of the invention are specifically embraced by the invention and disclosed herein as if each and every combination were individually and expressly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the invention and disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0022] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0023] Detailed Description The present invention provides a cargo delivery fusion polypeptide comprising an endosomolytic polypeptide and a cell penetrating polypeptide, and a composition comprising said cargo delivery fusion polypeptide. The present invention also provides a method of delivering a cargo to a target eukaryotic cell using a composition comprising an amphipathic polypeptide.

[0024] Cargo Delivery Fusion Polypeptides The present invention provides a cargo delivery fusion polypeptide comprising: a) an endosomolytic polypeptide; and b) a cell penetrating polypeptide (CPP). The cargo delivery fusion polypeptide of the present disclosure can be a polypeptide of any one of formulas I-V, as shown below. The cargo delivery fusion polypeptide of the present disclosure can be a polypeptide of any one of formulas VI-X, as shown below. The cargo delivery fusion polypeptide of the present disclosure is an amphipathic polypeptide that allows i) delivery of a polymeric cargo across a eukaryotic cell membrane, and / or ii) escape of the polymeric cargo from an endosome. The use of the cargo delivery fusion polypeptide of the present disclosure to deliver a cargo (e.g., a ribonucleoprotein (RNP) or other cargo) to a cell (in vitro or in vitro) is also referred to herein as "peptide-mediated delivery" or "PERC".

[0025] The cargo delivery fusion polypeptide of the present disclosure allows for the delivery of cargo to cells (e.g., eukaryotic cells), for example, the delivery of cargo to the cytoplasm of eukaryotic cells. The cargo delivery fusion polypeptide of the present disclosure allows for the delivery of cargo into eukaryotic cells, and the use of the cargo delivery fusion polypeptide to deliver cargo to eukaryotic cells is less toxic to cells than, for example, electroporation. For example, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more than 90% of the cell population contacted with the cargo delivery fusion polypeptide of the present disclosure remains viable for at least 48 hours after contact.

[0026] In some cases, the CPP portion of the cargo delivery fusion polypeptide of the present disclosure is an arginine-rich peptide having a length of about 8 amino acids to about 12 amino acids (e.g., 8, 9, 10, 11, or 12 amino acids). Examples of arginine-rich CPPs include, for example, YGRKKRRQRR (SEQ ID NO: 160), GRKKRRQRRR (SEQ ID NO: 161), GRKKRRQRR (SEQ ID NO: 162), and the like.

[0027] In some cases, the endosomolytic polypeptide present in the cargo delivery fusion polypeptide of the present disclosure is derived from an influenza hemagglutinin polypeptide. For example, in some cases, the endosomolytic polypeptide present in the cargo delivery fusion polypeptide of the present disclosure is derived from an E5 polypeptide (e.g., a peptide having the sequence: GLFEAIAEFIENGWEGLIEGWYG (SEQ ID NO: 163)). As another example, in some cases, the endosomolytic polypeptide present in the cargo delivery fusion polypeptide of the present disclosure is derived from an INF7 polypeptide (e.g., a peptide having the sequence: GLFEAIEGFIENGWEGMIDGWYG (SEQ ID NO: 164)). The endosomolytic polypeptide present in the cargo delivery fusion polypeptide of the present disclosure can have a length of 21 amino acids to 25 amino acids (e.g., 21, 22, 23, 24, or 25 amino acids).

[0028] The total length of a cargo delivery fusion polypeptide of the disclosure is from about 29 amino acids to about 37 amino acids (e.g., 29, 30, 31, 32, 33, 34, 35, 36, or 37 amino acids). In some cases, the total length of a cargo delivery fusion polypeptide of the disclosure is from about 32 amino acids to about 35 amino acids.

[0029] In some cases, a cargo delivery fusion polypeptide of the disclosure comprises a) an endosomolytic polypeptide and b) a cell penetrating polypeptide, wherein the fusion polypeptide has a length of about 32 amino acids to about 35 amino acids, any two adjacent amino acids are independently linked by an amide or non-amide bond, and the cargo delivery fusion polypeptide comprises one or more of i) a positively charged amino acid at the N-terminus, ii) a positively charged amino acid within 5 amino acids of the N-terminus, and iii) a positively charged amino acid at position 22. In some cases, the fusion polypeptide comprises the amino acid sequence of any one of Formulas I-VIII.

[0030] In some cases, the cargo delivery fusion polypeptide of the present disclosure comprises a positively charged amino acid as the N-terminal amino acid, for example, in some cases, the cargo delivery fusion polypeptide of the present disclosure comprises His or Lys as the N-terminal amino acid. In some cases, the cargo delivery fusion polypeptide comprises i) a positively charged amino acid as the N-terminal amino acid (e.g., comprises His or Lys as the N-terminal amino acid), and ii) a positively charged amino acid within 5 amino acids of the N-terminus. For example, the cargo delivery fusion polypeptide comprises i) a positively charged amino acid as the N-terminal amino acid (e.g., comprises His or Lys as the N-terminal amino acid), and ii) an Arg or Lys at amino acid 5. In some cases, the N-terminal amino acid is His and amino acid 5 is Arg. In some cases, the N-terminal amino acid is His and amino acid 5 is Lys. In some cases, the cargo delivery fusion polypeptide of the present disclosure comprises a positively charged amino acid at amino acid 22, for example, in some cases, the cargo delivery fusion polypeptide comprises Lys or Arg at position 22. In some cases, the cargo delivery fusion polypeptides of the disclosure include i) a positively charged amino acid as the N-terminal amino acid (e.g., His or Lys as the N-terminal amino acid), ii) a positively charged amino acid within 5 amino acids of the N-terminus (e.g., Arg or Lys at amino acid 5), and iii) a positively charged amino acid at amino acid 22 (e.g., Lys or Arg at amino acid 22).

[0031] In some cases, a cargo delivery fusion polypeptide of the disclosure is a polypeptide of formula I: KLFEX1IEGFIENGWEX2MIDX3WX4GX5GRKKRRQRR (SEQ ID NO: 165), where X1 is A, R, or K, X2 is A or G, X3 is L or G, X4 is N or Y, and X5, if present, is Y.

[0032] The polypeptide of formula I includes KLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 19, SEQ ID NO: 19), KLFEAIEGFIENGWEGMIDGWYGGRKKRRQRR (Peptide 40, SEQ ID NO: 40), KLFERIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 44, SEQ ID NO: 44), KLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 45, SEQ ID NO: 45), KLFEAIEGFIENGWEAMIDGWYGYGRKKRRQRR (Peptide 46, SEQ ID NO: 46), KLFEAIEGFIENGWEGMIDLWYGYGRKKRRQRR (Peptide 47, SEQ ID NO: 47), KLFEAIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 48, SEQ ID NO: 48), KLFEAIEGFIENGWEAMIDLWYGYGRKKRRQRR (Peptide 53, SEQ ID NO:53), KLFEAIEGFIENGWEAMIDGWNGYGRKKRRQRR (Peptide 54, SEQ ID NO:54), KLFEAIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 55, SEQ ID NO:55), KLFEAIEGFIENGWEAMIDLWNGYGRKKRRQRR (Peptide 56, SEQ ID NO:56), and KLFEKIEGFIENGWEAMIDLWNGYGRKKRRQRR (peptide 57, SEQ ID NO:57).

[0033] In some cases, a cargo delivery fusion polypeptide of the disclosure is a polypeptide of formula II: X1LFEX2IEGFIENGWEGMIDGWYGYGRKKRRQRR (SEQ ID NO: 166), where X1 is R or G and X2 is R or K.

[0034] The polypeptide of formula II includes RLFERIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 42, SEQ ID NO: 42); RLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 43, SEQ ID NO: 43), and GLFERIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 41, SEQ ID NO: 41).

[0035] In some cases, a cargo delivery fusion polypeptide of the disclosure is a polypeptide of formula III: GLFEAIEGFIENGWEX1MIDX2WNGYGRKKRRQRR (SEQ ID NO: 167), where X1 is A or G and X2 is G or L.

[0036] The polypeptide of formula III includes GLFEAIEGFIENGWEAMIDGWNGYGRKKRRQRR (Peptide 50, SEQ ID NO:50), GLFEAIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 51, SEQ ID NO:51), and GLFEAIEGFIENGWEAMIDLWNGYGRKKRRQRR (peptide 52, SEQ ID NO:52).

[0037] In some cases, a cargo delivery fusion polypeptide of the disclosure is a polypeptide of formula IV: GLFEAIEGFIENGWEX1X2IX3LWYGYGRKKRRQRR (SEQ ID NO: 168), where X1 is A or G, X2 is L or M, and X3 is D or E.

[0038] The polypeptide of formula V includes GLFEAIEGFIENGWEAMIDLWYGYGRKKRRQRR (Peptide 49, SEQ ID NO: 49), and GLFEAIEGFIENGWEGLIELWYGYGRKKRRQRR (peptide 58, SEQ ID NO:58).

[0039] In some cases, a cargo delivery fusion polypeptide of the disclosure is a polypeptide of formula V: GLFX1AIAX2FIX3NGWX4GLIX5GWYGGRKKRRQRRR (SEQ ID NO: 169), where each of X1, X2, X3, X4, and X5 is independently a non-coded amino acid.

[0040] In some cases, the non-encoded amino acid is α-aminoadipic acid. GLFαAIAαFIαNGWαGLIαGWYGGRKKRRQRRR (peptide 59, SEQ ID NO:59), or GLFαAIAαFIENGWEGLIDGWYGGRKKRRQRRR (Peptide 60, SEQ ID NO:60), where "α" is α-aminoadipic acid.

[0041] In some cases, a cargo delivery fusion polypeptide of the disclosure is a polypeptide of formula VI: KLFEX1IX2X3FIENGWEGMIX4X5WX6GYGRKKRRQRX7 (SEQ ID NO: 170), where X1 is A or H, X2 is E or A, X3 is G or E, X4 is D or E, X5 is G or L, X6 is E, H, K, R, or N, and X7, if present, is R.

[0042] Polypeptides of formula VI include KLFEAIEGFIENGWEGMIDLWEGYGRKKRRQRR (Peptide 62, SEQ ID NO: 62), KLFEAIEGFIENGWEGMIDLWHGYGRKKRRQRR (Peptide 63, SEQ ID NO: 63), KLFEAIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 64, SEQ ID NO: 64), KLFEAIEGFIENGWEGMIDLWRGYGRKKRRQRR (Peptide 65, SEQ ID NO: 65), KLFEAIEGFIENGWEGMIDLWNGYGRKKRRQR (Peptide 69, SEQ ID NO: 69), KLFEAIEGFIENGWEGMIELWNGYGRKKRRQRR (Peptide 71, SEQ ID NO: 71), KLFEAIAEFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 72, SEQ ID NO: 72), KLFEHIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 105, SEQ ID NO: 105), KLFEHIEGFIENGWEGMIDLWYGYGRKKRRQRR (peptide 107, SEQ ID NO: 107), and KLFEHIEGFIENGWEGMIDLWKGYGRKKRRQRR (peptide 109, SEQ ID NO: 109).

[0043] In some cases, a cargo delivery fusion polypeptide of the disclosure is a polypeptide of formula VII: GLFEX1IX2X3FIENGWEGMIDX4WX5GYGRKKRRQRR (SEQ ID NO: 171), where X1 is R, H, A, or K, X2 is E or A, X3 is G or E, X4 is L or G, and X5 is N, Y, K, or E.

[0044] Polypeptides of formula VII include GLFERIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 66, SEQ ID NO: 66); GLFEHIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 68, SEQ ID NO: 68); GLFEAIAEFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 70, SEQ ID NO: 70), GLFEKIEGFIENGWEAMIDGWYGYGRKKRRQRR (Peptide 73, SEQ ID NO: 73), GLFEKIEGFIENGWEGMIDLWYGYGRKKRRQRR (Peptide 74, SEQ ID NO: 74); GLFEAIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 75, SEQ ID NO: 31), GLFEAIEEFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 76, SEQ ID NO: 24), GLFEKIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 98, SEQ ID NO: 98); GLFEKIEGFIENGWEGMIDGWKGYGRKKRRQRR (Peptide 99, SEQ ID NO: 99); GLFEKIEGFIENGWEGMIDGWEGYGRKKRRQRR (Peptide 100, SEQ ID NO: 100), GLFEKIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 101, SEQ ID NO: 101), GLFEKIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 102, SEQ ID NO: 102), and GLFEKIEGFIENGWEGMIDLWEGYGRKKRRQRR (peptide 103, SEQ ID NO: 103).

[0045] In some cases, a cargo delivery fusion polypeptide of the disclosure is a polypeptide of Formula VIII: HLFEX1IEGFIENGWEGMIDX2WX3GYGRKKRRQRR (SEQ ID NO: 172), where X1 is A or K, X2 is G or L, and X3 is N, K, E, or Y.

[0046] Polypeptides of formula VIII include HLFEAIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 92, SEQ ID NO: 92), HLFEAIEGFIENGWEGMIDGWKGYGRKKRRQRR (Peptide 93, SEQ ID NO: 93), HLFEAIEGFIENGWEGMIDGWEGYGRKKRRQRR (Peptide 94, SEQ ID NO: 94), HLFEAIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 95, SEQ ID NO: 67), HLFEAIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 96, SEQ ID NO: 96), HLFEAIEGFIENGWEGMIDLWEGYGRKKRRQRR (Peptide 97, SEQ ID NO: 97), HLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 104, SEQ ID NO: 104), HLFEKIEGFIENGWEGMIDLWYGYGRKKRRQRR (Peptide 106, SEQ ID NO: 106), and HLFEKIEGFIENGWEGMIDLWKGYGRKKRRQRR (peptide 108, SEQ ID NO: 108).

[0047] In some cases, a fusion polypeptide of the disclosure comprises an amino acid sequence of Formula IX: KLFEAIEGFIENGWEGMIDLWNX1X2YGRKKRRQRR (SEQ ID NO: 173), where X1, if present, is Gly and X2 is Cys(methyltetrazine) or Cys(3-nitro-2-pyridinesulfenyl). In some cases, the fusion polypeptide comprises an amino acid sequence selected from KLFEAIGFIENGWEGMIDLWNC*YGRKKRRQRR (Peptide 87, SEQ ID NO:87) (where "C*" is Cys(methyltetrazine)), KLFEAIGFIENGWEGMIDLWNGC*YGRKKRRQRR (Peptide 88, SEQ ID NO:88) (where "C*" is Cys(methyltetrazine)), KLFEAIGFIENGWEGMIDLWNC*YGRKKRRQRR (Peptide 89, SEQ ID NO:89) (where "C*" is Cys(3-nitro-2-pyridinesulfenyl)), and KLFEAIGFIENGWEGMIDLWNGC*YGRKKRRQRR (Peptide 90, SEQ ID NO:90) (where "C*" is Cys(3-nitro-2-pyridinesulfenyl)).

[0048] In some cases, the fusion polypeptide of the disclosure may have the formula X:KLFEAIEGFIENGWEGMIDLWNGX1YGRKKRRQRRX2 (SEQ ID NO: 174), where X1 is Cys(methyltetrazine-PEG4-maleimide), Cys(maleimide), Lys(PEG 23)2, Lys(3-nitro-pyridine-2-carboxylic acid), Lys(PEG 23 )2, Lys(PEG 23 30 , wherein X2 is Cys(3-nitro-2-pyridine-sulfenyl) or Lys(methyltetrazine-PEG4). In some cases, the fusion polypeptide comprises an amino acid sequence selected from the amino acid sequences of peptide f1, peptide f2, peptide f3, peptide f4, peptide f4, peptide f6, peptide f7, peptide f11, peptide f13, and peptide f14 shown in FIG.

[0049] Non-limiting examples of suitable cargo delivery fusion polypeptides of the present disclosure include peptides no. 19 and no. 40-60 as shown in FIG.

[0050] Non-limiting examples of suitable cargo delivery fusion polypeptides of the disclosure include the polypeptides shown in FIG. 30 as peptide numbers 62-74, 76, 77, 87-109, and f1-f15.

[0051] In FIG. 30, the following comments regarding modified amino acids and / or non-amide bond linkages apply. i) {Ac} is an acetyl group; ii) {β-Ala} is beta-alanine; iii) {Ctz} refers to a cysteine ​​chemically modified with methyltetrazine-PEG4-maleimide; iv) {Cpy} refers to Cys(3-nitro-2-pyridinesulfenyl) incorporated into the peptide backbone; v) -PEG2- means that two copies (monomer units) of polyethylene glycol are incorporated into the peptide backbone; vi) -PEG4- means that four copies (monomer units) of polyethylene glycol are incorporated into the peptide backbone; vii) -PEG8- means that 8 copies (monomer units) of polyethylene glycol are incorporated into the peptide backbone; viii) {Kpi} refers to Lys(3-nitro-pyridine-2-carboxylic acid); ix) {Kp46} refers to Lys(PEG23)2, a branched PEG moiety extending from the peptide backbone; x) {Kp46py} refers to Lys(PEG23)2)-(3-nitro-pyridine-2-carboxylic acid) extended from the peptide backbone; xi) {Kfam} refers to Lys that has been chemically modified by conjugation with the succinimidyl ester form of 5-FAM (5-carboxyfluorescein); xii) {Cd} refers to a Cys that is chemically modified after synthesis with 1,4-bis(bromomethyl)-benzene to form a peptide dimer; and xiii) {Ktz} refers to Lys chemically modified by conjugation with methyltetrazine-PEG4-NHS ester.

[0052] In some cases, a cargo delivery fusion polypeptide of the disclosure does not include one or more of the amino acid sequences of the polypeptides depicted in FIG. 1 as peptides 1-18 and 20-37. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 1 depicted in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 2 depicted in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 3 depicted in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 4 depicted in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 5 depicted in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 6 depicted in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 7 depicted in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 8 depicted in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 9 depicted in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 10 depicted in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 11 shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 12 shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 13 shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 14 shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 15 shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 16 shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 17 shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 18 shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 20 shown in FIG. 1 is specifically excluded.In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 21 as shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 22 as shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 23 as shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 24 as shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 25 as shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 26 as shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 27 as shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 28 as shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 29 as shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 30 as shown in FIG. 1 is specifically excluded. In some cases, a fusion polypeptide comprising the amino acid sequence of peptide 31 as shown in FIG. 1 is specifically excluded. In some cases, fusion polypeptides comprising the amino acid sequence of peptide 32 shown in Figure 1 are specifically excluded. In some cases, fusion polypeptides comprising the amino acid sequence of peptide 33 shown in Figure 1 are specifically excluded. In some cases, fusion polypeptides comprising the amino acid sequence of peptide 34 shown in Figure 1 are specifically excluded. In some cases, fusion polypeptides comprising the amino acid sequence of peptide 35 shown in Figure 1 are specifically excluded. In some cases, fusion polypeptides comprising the amino acid sequence of peptide 36 shown in Figure 1 are specifically excluded. In some cases, fusion polypeptides comprising the amino acid sequence of peptide 37 shown in Figure 1 are specifically excluded.

[0053] In some cases, one or more of the peptides depicted in peptides 78-86 of FIG. 30 are specifically excluded. In some cases, a polypeptide comprising the amino acid sequence LFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR (SEQ ID NO: 175) is specifically excluded. In some cases, a polypeptide comprising the amino acid sequence {β-Ala}LFEAIGFIENGWEGMIDGWYGYGRKKRRQRR (SEQ ID NO: 78), where {β-Ala} is an acetyl group at the N-terminus of the polypeptide, is specifically excluded. In some cases, a polypeptide comprising the amino acid sequence GLFEEIEGFIENGWEGMIDGWYGYGRKKRRQRR (SEQ ID NO: 79) is specifically excluded. In some cases, a polypeptide comprising the amino acid sequence GLFEAIEGFIENEWEGMIDGWYGYGRKKRRQRR (SEQ ID NO: 80) is specifically excluded. In some cases, a polypeptide comprising the amino acid sequence GLFEAIEGFIENGWEGMIEGWYGYGRKKRRQRR (SEQ ID NO: 81) is specifically excluded. In some cases, a polypeptide comprising the amino acid sequence GLFEAIEGFIENGWEGMIDGWYGYGHKKHHQHH (SEQ ID NO: 82) is specifically excluded. In some cases, a polypeptide comprising the amino acid sequence GLFEAIEGFIENGWEGMIDGWYGYGRKKRRQR (SEQ ID NO: 32) is specifically excluded. In some cases, a polypeptide comprising the amino acid sequence GLFEAIEGFIENGWEGMIDGWYGYGRKKRRQ (SEQ ID NO: 84) is specifically excluded. In some cases, a polypeptide comprising the amino acid sequence GLFEAIEGFIENGWEGMIDGWYGYGRKKRR (SEQ ID NO: 85) is specifically excluded. In some cases, a polypeptide comprising the amino acid sequence GLFEAIEGFIENGWEGMIDGWYGYGHKKHHQHR (SEQ ID NO: 33) is specifically excluded.

[0054] Modified Amino Acids The cargo-delivering fusion polypeptides of the present disclosure can include one or more modified amino acids. In some cases, the amino acids present in the cargo-delivering fusion polypeptides include modifications ("functional moieties") that allow for linkage to a second polypeptide or other moiety. Suitable modifications include thiol-reactive moieties, amine-reactive moieties, haloacetyl groups (e.g., iodoacetamide, chloroacetamide, etc.), and members of click chemistry pairs. For example, thiol-reactive groups include, for example, haloacetyl, maleimide, aziridine, acryloyl, arylating agents, vinyl sulfone, and pyridyl disulfide. Click chemistry pairs include, for example, i) azide and dibenzocyclooctyne, and ii) tetrazine and trans-cyclooctene.

[0055] Functional groups that allow for conjugation include, but are not limited to, azide groups, alkynyl groups, phosphine groups, cysteine ​​residues, C-terminal thioesters, aryl azides, maleimides, carbodiimides, N-hydroxysuccinimide (NHS) esters, hydrazides, PFP esters, hydroxymethylphosphine, psoralens, imidoesters, pyridyl disulfides, isocyanates, aminooxy, aldehydes, keto, chloroacetyl, bromoacetyl, and vinyl sulfones. Suitable functional groups include, for example, isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters.

[0056] Examples of cargo delivery fusion polypeptides of the disclosure include, for example, peptides designated as peptide number 77, peptide number 87, peptide number 88, peptide number 89, peptide number 90, peptide number 91, peptide number f4, peptide number f5, peptide number f6, peptide number f7, peptide number f8, peptide number f9, peptide number f10, peptide number f11, peptide number f12, peptide number f13, peptide number f14, and peptide number f15, as shown in FIG.

[0057] Non-amide bond linkage In some cases, the amino acids of the cargo delivery fusion polypeptide of the disclosure are all linked by amide bonds. In some cases, the cargo delivery fusion polypeptide of the disclosure includes one or more linkages other than amide bonds. For example, in some cases, the cargo delivery fusion polypeptide includes one or more PEG moieties in place of an amide bond between two adjacent amino acids. In some cases, the cargo delivery fusion polypeptide includes a single PEG moiety (e.g., a PEG2 moiety, a PEG4 moiety, a PEG8 moiety, etc.) in place of an amide bond between two adjacent amino acids. Suitable PEG moieties include PEG moieties having 2-8 ethylene glycol units. For example, in some cases, the cargo delivery fusion polypeptide includes a PEG2 linkage. As another example, in some cases, the cargo delivery fusion polypeptide includes a PEG4 linkage. As another example, in some cases, the cargo delivery fusion polypeptide includes a PEG8 linkage. A non-limiting example is shown in FIG. 30.

[0058] composition The present disclosure provides a composition comprising the cargo delivery fusion polypeptide of the present disclosure. The composition of the present disclosure may contain, in addition to the cargo delivery fusion polypeptide of the present disclosure, salts such as NaCl, MgCl2, KCl, MgSO4, etc.; buffers such as 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 ( the like; solubilizing agents; protease inhibitors; detergents, such as Tween-20, Tween-80, etc.; nuclease inhibitors; glycerol; reducing agents (e.g., dithiothreitol (DTT)); solubilizing agents (e.g., dimethylsulfoxide (DMSO)); non-ionic detergents; synthetic polymers, etc.

[0059] In some cases, the composition of the present disclosure comprises a) a cargo delivery fusion polypeptide of the present disclosure and b) DMSO. In some cases, the cargo delivery fusion polypeptide of the present disclosure is maintained in a solution comprising DMSO at a concentration of about 9% DMSO to about 15% DMSO (e.g., about 10% DMSO) for a period of time before being contacted with the cargo. If the cargo delivery fusion polypeptide is prepared in a DMSO solution of less than 10%, the peptides may bind to each other and in some cases may not effectively bind to the cargo. Thus, in some cases, the cargo delivery fusion polypeptide is kept in a solution of about 10% DMSO for a period of time, and then the peptide is contacted with the cargo present in a solution that does not contain DMSO (e.g., a buffered aqueous solution). Thus, the cargo delivery fusion polypeptide / cargo solution may comprise 1% DMSO to 5% DMSO. In some cases, the cargo delivery fusion polypeptide / cargo solution comprises less than 1% DMSO, for example, in some cases, the cargo delivery fusion polypeptide / cargo solution comprises 0.01% to 1% DMSO.

[0060] In some cases, a composition of the present disclosure comprises a) a cargo-delivering fusion polypeptide of the present disclosure, and b) an organic solvent other than DMSO. Suitable organic solvents include, for example, ethanol, methanol, dimethylformamide, gamma butyrolactone, N-methyl-2-pyrrolidone, and dimethylacetamide.

[0061] In some cases, the composition of the present disclosure comprises a) a cargo-delivering fusion polypeptide of the present disclosure and b) poly(ethylene glycol) (PEG). In some cases, the composition of the present disclosure comprises a) a cargo-delivering fusion polypeptide of the present disclosure and b) a non-ionic surfactant. Suitable non-ionic surfactants include, for example, polysorbate 80, polyoxyethylene (23) lauryl ether (brij-L23), poloxamer (i.e., a non-ionic triblock copolymer consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide))).

[0062] In some cases, the composition of the present disclosure comprises a) a cargo-delivering fusion polypeptide of the present disclosure and b) saline (e.g., 0.9% NaCl). In some cases, the composition is sterile. In some cases, the pharmaceutical composition of the present invention is suitable for administration to a human subject, e.g., the composition is sterile and free of detectable pyrogens and / or other toxins. Thus, the present disclosure provides a composition comprising a) a cargo-delivering fusion polypeptide of the present disclosure and b) saline (e.g., 0.9% NaCl), the composition being sterile and free of detectable pyrogens and / or other toxins. In some cases, the composition further comprises a cargo to be delivered.

[0063] In some cases, the composition of the present disclosure comprises a) a cargo delivery fusion polypeptide of the present disclosure, and b) a cargo to be delivered to a eukaryotic cell. Any of a variety of cargoes can be included in the composition of the present disclosure. In some cases, the cargo is a nucleic acid. In some cases, the cargo is a nucleic acid comprising a nucleotide sequence encoding a gene product of interest. In some cases, the cargo is a polypeptide. In some cases, the cargo is a CRISPR-Cas effector polypeptide, or a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide. In some cases, the cargo is a ribonucleoprotein (RNP) comprising i) a CRISPR-Cas effector polypeptide, and ii) a CRISPR-Cas guide nucleic acid. In some cases, the cargo is an RNP comprising i) a CRISPR-Cas effector polypeptide, and ii) a guide nucleic acid comprising an activation segment comprising a nucleotide sequence that binds to the CRISPR-Cas effector polypeptide and a targeting segment comprising a nucleotide sequence that hybridizes to a target nucleic acid. In some cases, the cargo is a DNA molecule that includes a nucleotide sequence encoding both a CRISPR-Cas effector protein and a CRISPR-Cas guide RNA. In some cases, the cargo is composed of i) an mRNA encoding a CRISPR-Cas effector polypeptide and ii) a CRISPR-Cas guide nucleic acid (e.g., a CRISPR-Cas guide RNA such as a single molecule guide RNA (sgRNA)).

[0064] In some cases, the cargo is not covalently linked to the cargo-delivery fusion polypeptide. In some cases, the cargo is covalently linked to the cargo-delivery fusion polypeptide, either directly or indirectly (e.g., indirectly via a linker).

[0065] In some cases, the cargo delivery fusion polypeptide is present in the composition at a concentration of about 2 μM to about 50 μM. For example, in some cases, the cargo delivery fusion polypeptide is present in the composition at a concentration of about 2 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 30 μM, about 30 μM to about 40 μM, or about 40 μM to about 50 μM.

[0066] Nucleic Acid Cargo As mentioned above, in some cases, the cargo is a nucleic acid. Thus, in some cases, the composition of the present disclosure comprises a) a cargo delivery fusion polypeptide of the present disclosure, and b) a nucleic acid cargo to be delivered to a eukaryotic cell. In some cases, the cargo is a nucleic acid comprising a nucleotide sequence encoding a gene product of interest ("cargo gene product"). The gene product of interest includes nucleic acids and polypeptides. For example, in some cases, the cargo is a DNA molecule comprising a nucleotide sequence encoding a gene product of interest ("cargo nucleotide sequence"). In some cases, the cargo comprises two or more DNA molecules each comprising a nucleotide sequence encoding a different gene product of interest. In some cases, the cargo is a DNA molecule comprising a nucleotide sequence encoding two or more gene products of interest.

[0067] In some cases, the cargo nucleotide sequence encoding the gene product of interest is operably linked to one or more transcriptional control elements, such as a promoter. In some cases, the cargo nucleotide sequence encoding the gene product of interest is operably linked to a promoter that functions in a selected cell type (e.g., eukaryotic cells, plant cells, animal cells, mammalian cells, primate cells, rodent cells, human cells, neurons, dendritic cells, epithelial cells, T cells, natural killer (NK) cells, hematopoietic stem cells, etc.).

[0068] In some cases, the promoter is a constitutively active promoter. In some cases, the promoter is a regulatable promoter. In some embodiments, the promoter is an inducible promoter. In some cases, the promoter is a tissue-specific promoter. In some cases, the promoter is a cell type-specific promoter. In some cases, the transcriptional control element (such as a promoter) functions in a target cell type or target cell population.

[0069] In some cases, the cargo nucleotide sequence encodes an RNA gene product. The RNA gene product of interest includes, for example, an inhibitory RNA, a CRISPR-Cas guide RNA, and the like. The RNA gene product of interest includes an RNA that inhibits or reduces the production of a harmful or otherwise undesirable protein.

[0070] In some cases, the cargo nucleotide sequence encodes a cargo polypeptide. Cargo polypeptide gene products of interest include, for example, therapeutic polypeptides, immunogenic polypeptides, growth factors, cytokines, enzymes, anti-angiogenic polypeptides, soluble receptors, antibodies, synthetic polypeptides (e.g., chimeric antigen receptors), blood clotting factors, peptide hormones, and the like.

[0071] In some cases, the cargo nucleotide sequence encoding the cargo polypeptide of interest is operably linked to a nucleotide sequence encoding a secretory signal peptide (e.g., 5'-ATGAAGATCATCCTGTGGCTGTGTGTTCGGCCTGTTCCTGGCCACCATGTTCCCCATCAGCTGGCAGATGCCCGTGGAGTCGGCCTGTCCTCCGAGGACTCCGCCAGCTCCGAGAGCTTCGCC-3' (SEQ ID NO: 176); Jeong et al (2012) J. Control Release 159(3):368-75). In some cases, a nucleotide sequence encoding a furin cleavage site (RGRR) is inserted between the nucleotide sequence encoding the secretory signal peptide and the nucleotide sequence encoding the cargo polypeptide of interest. The secretory signal peptide of the expressed protein is removed in the Golgi apparatus, and the polypeptide of interest is secreted.

[0072] In some cases, the cargo nucleic acid comprises a nucleotide sequence encoding an antibody. In some cases, the encoded antibody is a therapeutic antibody. In some cases, the nucleotide sequence encoding the antibody comprises a nucleotide sequence encoding a secretory signal peptide as described above, such that the therapeutic polypeptide is secreted extracellularly upon expression of the cargo in the target cell.

[0073] Targeting antibodies In some cases, the cargo comprises a targeting antibody. The targeting antibody can be linked to the cargo (e.g., the RNP containing a CRISPR Cas effector polypeptide or other gene editing mechanism), and the linkage (e.g., covalent linkage) can be direct or via a linker. In some cases, the linker is a proteolytically cleavable linker. The targeting antibody can be specific for a cell surface protein on the surface of the cell ("target cell") to which the cargo is delivered. In some cases, the cell surface protein targeted by the targeting antibody is a cancer-associated antigen (e.g., EpCAM, E-cadherin, EMA, HER2 / neu, alpha-fetoprotein, beta-hCG, bladder tumor antigen, BCR-ABL, CEA, CD19, CD22, CD30, MCAM (Muc18), metadherin, glypigan 2, PSMA, human transferrin receptor, EGRF complex, AXL, PTK7, etc.), a specific cell type receptor (e.g., CD4, CD8, TCR for T cells), a stem cell marker (e.g., TRA-1-60, TRA-1-81, SSEA), or a lineage marker (e.g., CD14 (monocytes), CD16 (NK cells, granulocytes), CD19 (B lymphocytes), CD20 (B lymphocytes), and CD56 (NK cells) for humans). In some cases, targeting of the cargo is achieved using an aptamer, a small molecule, a peptide, a toxin, a carbohydrate, a vitamin, or another targeting moiety, such as transferrin.

[0074] Polypeptide Cargo As mentioned above, in some cases, the cargo is a polypeptide. Thus, in some cases, the composition of the present disclosure comprises a) the cargo delivery fusion polypeptide of the present disclosure, and b) the polypeptide cargo to be delivered to eukaryotic cells. The cargo polypeptide of interest can be, for example, a therapeutic polypeptide, an immunogenic polypeptide, a growth factor, a cytokine, an enzyme, an antiangiogenic polypeptide, a soluble receptor, an antibody, a synthetic polypeptide (e.g., a chimeric antigen receptor), a blood clotting factor, a peptide hormone, etc.

[0075] In some cases, the cargo polypeptide comprises a targeting moiety that aids in delivery of the cargo to a desired cell or tissue type. In some cases, the targeting moiety is an antibody or fragment thereof, a DARPin, an aptamer, or the like.

[0076] antibody In some cases, the cargo polypeptide is an antibody. Thus, in some cases, the composition of the present disclosure comprises a) a cargo delivery fusion polypeptide of the present disclosure and b) an antibody to be delivered to a eukaryotic cell. Suitable antibodies are described elsewhere herein. The antibody may be any antigen-binding antibody-based polypeptide, a variety of which are known in the art. In some cases, the antibody is a single chain Fv (scFv). Other antibody-based recognition domains suitable for use include cAb VHH (camelid antibody variable domain) and humanized versions, IgNAR VH (shark antibody variable domain) and humanized versions, sdAb VH (single domain antibody variable domain), and "camelized" antibody variable domains. In some cases, the cargo polypeptide comprises a T cell receptor (TCR)-based recognition domain, such as a single chain TCR (scTv, single chain two domain TCR including VαVβ). In some cases, the cargo antibody is a therapeutic antibody.

[0077] The antibody may be specific for an antigen such as CD49f, CD34, CD90, CD117, CXCR4, CD79, CD22, RP105, CD71, CD28, CD94 (KLDR1), CD56, XCR1, CD205, CD370, CD209, CD54, CD335, NCR1, CD94, NKG2D, NKp30, CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, etc. In some cases, the antibody is specific for a cytokine. In some cases, the antibody is specific for a cytokine receptor. In some cases, the antibody is specific for a growth factor. In some cases, the antibody is specific for a growth factor receptor. In some cases, the antibody is specific for a cell surface receptor. In some cases, the antibody is an anti-CD3 antibody.

[0078] Immunogenic Polypeptides In some cases, the cargo is an immunogenic polypeptide. The immunogenic protein is suitable for stimulating an immune response against an antigenic protein in a mammalian host (e.g., human, non-human primate, bovine (e.g., cow), ovine (e.g., sheep), equine (e.g., horse), porcine (e.g., pig), etc.). The immunogenic polypeptide can be derived from a self-antigen, an allergen, a tumor-associated antigen, a pathogenic virus, a pathogenic bacterium, a pathogenic protozoan, a pathogenic helminth, or other pathogenic organism that infects a mammalian host.

[0079] The viral antigen may be any antigen of a variety of viral pathogens.

[0080] In other embodiments, the viral pathogen is an Adenoviridae, Arenaviridae, Astroviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Hepeviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. In some cases, the virus is adenovirus, coronavirus, coxsackievirus, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 2, cytomegalovirus, human herpesvirus type 8, human immunodeficiency virus, influenza virus, measles virus, mumps virus, human papillomavirus, parainfluenza virus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, or varicella zoster virus. Viruses include those from the Adenoviridae family (e.g., adenovirus), Arenaviridae family (e.g., Machupo virus), Bunyaviridae family (e.g., Hantavirus or Rift Valley fever virus), Coronaviridae family, Orthomyxoviridae (e.g., influenza virus), Filoviridae (e.g., Ebola virus and Marburg virus), Flaviviridae (e.g., Japanese encephalitis virus and yellow fever virus), Hepadnaviridae (e.g., hepatitis B virus), Herpesviridae (e.g., herpes simplex virus), Papovaviridae (e.g., papilloma virus), and the like. The virus may be selected from the following families: Paramyxoviridae (e.g., Hausatorum Syncytial Virus, Measles Virus, Mumps Virus, or Parainfluenza Virus), Parvoviridae, Picornaviridae (e.g., Poliovirus), Poxviridae (e.g., Smallpox Virus), Reoviridae (e.g., Rotavirus), Retroviridae (e.g., Human T-cell Lymphotropic Virus (HTLV) and Human Immunodeficiency Virus (HIV)), Rhabdoviridae (such as rabies virus), and Togaviridae (such as encephalitis virus, yellow fever virus, and rubella virus).

[0081] In some cases, viral antigens may be Adenovirus, Alphavirus (Togavirus), Eastern Equine Encephalitis Virus, Eastern Equine Encephalomyelitis Virus, Venezuelan Equine Encephalomyelitis Vaccine Strain TC-83, Western Equine Encephalomyelitis Virus, Arenavirus, Lymphocytic Choriomeningitis Virus (non-neurotropic strains), Tacaribe Virus Complex, Bunyavirus, Bunyamwera Virus, Rift Valley Fever Virus Vaccine Strain MP-12, Calcivirus, Coronavirus, Flavivirus (Togavirus) - Group B Arbovirus, Dengue Virus Serotypes 1, 2, 3, 4, Yellow Virus, and others. Fever virus vaccine strain 17D, hepatitis A, B, C, D, E viruses, cytomegalovirus, Epstein-Barr virus, herpes simplex types 1 and 2, varicella zoster, human herpesviruses 6 and 7, influenza viruses A, B, C, papovavirus, papillomavirus, Newcastle disease virus, measles virus, mumps virus, parainfluenza viruses 1, 2, 3, 4, polyomavirus (JC virus, BK virus), respiratory syncytial virus, human parvovirus (B19), coxsackievirus A types A and B, echovirus, poliovirus, rhinovirus, alastrim (variola minor virus), smallpox (variola major virus), whitepox reovirus, coltivirus, human rotavirus, and orbivirus (Colorado tick fever virus), rabies virus, vesicular stomatitis virus, rubivirus (rubella), Semliki Forest virus, Saint Louis encephalitis virus, Venezuelan equine encephalomyelitis virus, arenavirus (also known as South American hemorrhagic fever virus), flexa, lymphocytic choriomeningitis virus (LCM) (neurotropic strains), Hantaan virus, Hantaviruses including HIV, Rift Valley fever virus, Japanese encephalitis virus, Yellow fever virus, Monkeypox virus, Human immunodeficiency virus (HIV) types 1 and 2, Human T-cell lymphotropic virus (HTLV) types 1 and 2, Simian immunodeficiency virus (SIV), Vesicular stomatitis virus, Guanarito virus, Lassa fever virus, Junin virus, Machupo virus, Sabia virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, Tick-borne encephalitis virus complex (Flavivirus), including Central European tick-borne encephalitis, Far Eastern tick-borne encephalitis,Hanzalova, Hipur, Kumringe, Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian spring-summer encephalitis viruses, Herpesvirus Simia (Herpes B or Monkey B virus), Cercopithecian herpesvirus 1 (Herpes B virus), Equine morbillivirus (Hendra virus and Hendra-like virus), Nipah virus, Variola major virus (Smallpox virus), Variola minor virus (Alastrim), African swine fever virus, African horse sickness virus, Akabane virus, Avian influenza virus (highly pathogenic), Bluetongue virus, Camelpox virus, Classical swine fever virus, Cowdria ruminantium (Cowdria ruminantium (heartwater disease), foot and mouth disease virus, goat pox virus, Japanese encephalitis virus, lumpy skin disease virus, malignant catarrhal fever virus, Menangle virus, Newcastle disease virus (VVND), peste des petits ruminants virus, rinderpest virus, sheep pox virus, swine vesicular disease virus, and vesicular stomatitis virus.

[0082] In some cases, the bacterial antigen may be selected from the group consisting of Bacillus (e.g., B. anthracis), Enterobacteriaceae (e.g., Salmonella, Escherichia coli, Yersinia pestis, pestis, Klebsiella, and Shigella), Yersinia (e.g., Y. pestis or Y. enterocolitica), Staphylococcus (e.g., S. aureus), Streptococcus, Neisseria gonorrhoeae, Enterococcus (e.g., E. faecalis), Listeria (e.g., L. monocytogenes), Brucella (e.g., B. abortus, B. melitensis, or B. suis), Vibrio (e.g., V. cholerae), Corynebacterium diphtheria, Pseudomonas (e.g., P. pseudomallei or P. aeruginosa), Burkholderia (e.g., B. mallei or B. pseudomallei), Shigella (e.g., S. dysenteriae), Rickettsia (e.g., R. rickettsii, R. prorowazekii, or R. typhi), Francisella tularensis, Chlamydia psittaci, Coxiella burnetii, Mycoplasma (e.g., M. mycoides).

[0083] In some cases, the bacterial antigen may be Acinetobacter baumannii (formerly Acinetobacter calcoaceticus), Actinobacillus spp., Actinomyces pyogenes (formerly Corynebacterium pyogenes), Actinomyces israelii, Nocardia asteroides, N. brasiliensis, Aeromonas hydrophila, Amycolata autotrophica, Archanobacterium haemolyticum (formerly Corynebacterium haemolyticum), Arizona hinshawii (all serotypes), Bacillus anthracis, Bacteroides fragilis, Bartonella henselae, B. quintana, B. vinsonii, Bordetella spp. (including B. pertussis), Borrelia recurrentis, B. burgdorferi, Burkholderia spp. (formerly Pseudomonas spp.), Campylobacter coli, coli), C. fetus, C. jejuni, Chlamydia psittaci, C. trachomatis, C. pneumonia, Clostridium botulinum (neurotoxin producing species), Cl. chauvoei, Cl. haemolyticum, Cl. histolyticum, Cl. novyinovyi, Cl. septicum, Cl. tetani, Cl. perfringens, Corynebacterium diphtheriae, C. pseudotuberculosis, C. renale, Dermatophilus congolensis, Edwardsiella tarda, Erysipelothrix rhusiopathiae, Escherichia coli (all enteropathogenic, enterotoxin-producing, enteroinvasive, and K1 antigen-containing strains (including E. coli O157:H7)), Haemophilus ducreyi, H. influenzae, Helicobacter pylori pylori, Klebsiella spp. (all species), Legionella spp. (including L. pneumophila), pathogenic Leptospira interrogans (all serotypes), Listeria spp., Moraxella spp., Mycobacterium spp. (MAC bacteria (M. avium) complex, M. asiaticum, M. bovis BCG vaccine strain, M. chelonei, M. fortuitum, M. kansasii, M. leprae, M. malmoense, M. marinum, M. paratuberculosis, M. scrofulaceum, M. simiae, M. szulgai, M. ulcerans, M. xenopi), Mycoplasma spp., Neisseria gonorrhoeae gonorrhoeae, N. meningitides, Nocardia asteroides, N. braziliensis, N. otitidiscaviarum, N. transvalensis, Proteus mirabilis, P. vulgarisvulgaris, Rhodococcus equi, Salmonella spp. (including S. arizonae, S. cholerasuis, S. enteritidis, S. gallinarum-pullorum, S. meleagridis, S. paratyphi A, B, C, S. typhi, S. typhimurium), Shigella spp. (including S. boydii, S. dysenteriae type 1, S. flexneri, S. sonnei), Sphaerophorus necrophorus necrophorus, Staphylococcus aureus, Streptobacillus moniliformis, Streptococcus spp. (including S. pneumoniae and Streptococcus pyogenes), Treponema pallidum, T. carateum, Vibrio cholerae, V. parahemolyticus, V. vulnificus, Yersinia enterocolitica enterocolitica, Bartonella spp., Brucella spp. (including B. abortus, B. canis, B. suis, B. melitensis), Burkholderia spp., Pseudomonas mallei, B. pseudomallei, Coxiella spp., Francis tularensis, Mycobacterium bovis, M. tuberculosis, Mycobacteria, Pasteurella multocida type B ("Buffalo" and other pathogenic strains), Rickettsia akari, akari), R. australis, R. canada, R. conorii, Rickettsia typhi, R. rickettsii, R. siberica, R.Antigens of bacterial pathogens selected from R. tsutsugamushi, R. mooseri, and Yersinia pestis.

[0084] In some cases, the antigen is a protozoan antigen, such as an antigen from a protozoan, such as Cryptosporidium parvum, Encephalitozoon, Plasmodium (e.g., Plasmodium falciparum), Toxoplasma gondii, Acanthamoeba, Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, Leishmania, or Trypanosoma (e.g., T. brucei, T. cruzi).

[0085] In some cases, the immunogenic polypeptide is a cancer-associated antigen, including, but not limited to, CD19, CD22, MUC1 polypeptide, human papillomavirus (HPV) E6 polypeptide, LMP2 polypeptide, HPV E7 polypeptide, epidermal growth factor receptor (EGFR) vIII polypeptide, HER-2 / neu polypeptide, melanoma antigen family A, 3 (MAGEA3) polypeptide, p53 polypeptide, mutant p53 polypeptide, NY-ESO-1 polypeptide, folate hydrolase (prostate-specific membrane antigen, PSMA) polypeptide, carcinoembryonic antigen (CEA) polypeptide, melanoma antigen recognized by T cells (melanA / MART1) polypeptide, Ras polypeptide, gp100 polypeptide, proteinase 3 (PR1) polypeptide, bcr-abl polypeptide, tyrosinase polypeptide, survivin polypeptide, prostate-specific antigen (PSA) polypeptide, hTERT polypeptide, sarcoma translocation breakpoint polypeptide, synovial sarcoma X (SSX) breakpoint polypeptide, EphA2 polypeptide, acid phosphatase, prostate (PAP) polypeptide, melanoma inhibitor of apoptosis (ML-IAP) polypeptide, alpha-fetoprotein (AFP) polypeptide, epithelial cell adhesion molecule (EpCAM) polypeptide, ERG (TMPRSS2 ETS fusion) polypeptide, NA17 polypeptide, paired box 3 (PAX3) polypeptide, anaplastic lymphoma kinase (ALK) polypeptide, androgen receptor polypeptide, cyclin B1 polypeptide, N-myc proto-oncogene (MYCN) polypeptide, Ras homolog gene family member C (RhoC) polypeptide, tyrosinase-related protein-2 (TRP-2) polypeptide, mesothelin polypeptide, prostate stem cell antigen (PSCA) polypeptide, melanoma-associated antigen-1 (MAGEA1) polypeptide, cytochrome P4501B1 (CYP1B1) polypeptide, placenta-specific protein 1 (PLAC1) polypeptide, BORIS polypeptide (also known as CCCTC-binding factor or CTCF), ETV6-AML polypeptide, breast cancer antigen NY-BR-1 polypeptide (also known as ankyrin repeat domain-containing protein 30A), regulator of G protein signaling (RGS5) polypeptide, squamous cell carcinoma antigen recognized by T cells (SART3) polypeptide, carbonic anhydrase IX polypeptide, paired box 5 (PAX5) polypeptide, OY-TES1 (testis antigen; also known as acrosin-binding protein) polypeptide, sperm protein 17 polypeptide, lymphoid cell-specific protein tyrosine kinase (LCK) polypeptide, high molecular weight melanoma-associated antigen (HMW-MAA), A-kinase anchoring protein-4 (AKAP-4), synovial sarcoma X breakpoint 2 (SSX2) polypeptide, X antigen family member 1 (XAGE1) polypeptide, B7 homolog 3 (B7H3, also known as CD276) polypeptide, legumain polypeptide (LGMN1, also known as asparaginyl endopeptidase), tyrosine kinase-2 with Ig and EGF homology domains (Tie-2, also known as angiopoietin-1 receptor) polypeptide, P antigen family member 4 (PAGE4) polypeptide, vascular endothelial growth factor receptor 2 (VEGF2) polypeptide, MAD-CT-1 polypeptide, fibroblast activation protein (FAP) polypeptide, platelet-derived growth factor receptor beta (PDGFβ) polypeptide, MAD-CT-2 polypeptide, Fos-related antigen 1 (FOSL) polypeptide, or Wilms' tumor 1 (WT-1) polypeptide.

[0086] CRISPR-Cas effector polypeptides In some cases, the cargo is a CRISPR-Cas effector polypeptide. The CRISPR-Cas effector polypeptide suitable for inclusion in the composition of the present disclosure is a class 2 CRISPR effector polypeptide, also referred to herein as class 2 CRISPR-Cas effector polypeptide. For example, in some cases, the CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide. In some cases, the type II CRISPR-Cas effector polypeptide is a Cas9 polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a type V CRISPR-Cas effector polypeptide, such as Cas12a, Cas12b, Cas12c, Cas12d, or Cas12e polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a type VI CRISPR-Cas effector polypeptide, such as Cas13a polypeptide, Cas13b polypeptide, Cas13c polypeptide, or Cas13d polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a Cas14 polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a Cas14a polypeptide, a Cas14b polypeptide, or a Cas14c polypeptide. CRISPR-Cas effector polypeptides suitable for inclusion in the compositions of the present disclosure include CRISPRi polypeptides (see, for example, Qi et al. (2013) Cell 152:1173; and Jensen et al. (2021) Genome Research doi:10.1101 / gr.275607.121.). CRISPR-Cas effector polypeptides suitable for inclusion in the compositions of the present disclosure include CRISPRa polypeptides (see, e.g., Jensen et al. (2021) Genome Research doi:10.1101 / gr.275607.121; and Breinig et al. (2019) Nature Methods 16:51.).CRISPR-Cas effector polypeptides suitable for inclusion in the compositions of the present disclosure include CRISPRoff polypeptides (see, e.g., Nunez et al. (2021) Cell 184:2503.). CRISPR-Cas effector polypeptides suitable for inclusion in the compositions of the present disclosure include nickases. CRISPR-Cas effector polypeptides suitable for inclusion in the compositions of the present disclosure include catalytically inactive CRISPR-Cas effector polypeptides that retain binding to a target nucleic acid (when complexed with a guide RNA). CRISPR-Cas effector polypeptides suitable for inclusion in the compositions of the present disclosure include fusion polypeptides that include i) a CRISPR-Cas effector polypeptide and ii) one or more heterologous fusion partners (also referred to as "heterologous polypeptides").

[0087] In some cases, a CRISPR-Cas effector polypeptide suitable for inclusion in a composition of the present disclosure is a Cas9 polypeptide. In some cases, the Cas9 polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more than 99% amino acid sequence identity to the Cas9 of Streptococcus pyogenes shown in Figure 28A.

[0088] In some cases, the Cas9 polypeptide is a Staphylococcus aureus Cas9 (saCas9) polypeptide. In some cases, the saCas9 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any known saCas9 amino acid sequence, such as the saCas9 amino acid sequence shown in Figure 28B.

[0089] In some cases, a suitable Cas9 polypeptide is a high fidelity (HF) Cas9 polypeptide (Kleinstiver et al. (2016) Nature 529:490.). For example, amino acids N497, R661, Q695, and Q926 of the amino acid sequence depicted in FIG. 28A are substituted, e.g., with alanine. For example, a HF Cas9 polypeptide can include an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in FIG. 28A, where amino acids N497, R661, Q695, and Q926 are substituted, e.g., with alanine. In some cases, a suitable Cas9 polypeptide exhibits altered PAM specificity (see, e.g., Kleinstiver et al. Nature (2015) 529:481).

[0090] In some cases, a suitable Cas9 polypeptide comprises an R691A substitution. For example, in some cases, a suitable Cas9 polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 28A, where amino acid 691 is Ala.

[0091] In some cases, a suitable Cas9 polypeptide comprises the following substitutions: D1135V, R1335Q, and T1337R. For example, in some cases, a suitable Cas9 polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 28A, where amino acid 1135 is Val, amino acid 1335 is Gln, and amino acid 1337 is Arg, and the Cas9 polypeptide exhibits relaxed PAM requirements.

[0092] In some cases, a suitable Cas9 polypeptide is a SpRY variant. See, for example, Zhang and Zhang (2020) Trends Genetics 36:546, and Walton et al. (2020) Science 368:290, and US Patent Publication No. 2021 / 0284978. SpRY is a variant of Streptococcus pyogenes Cas9, which has relaxed PAM requirements. For example, in some cases, a suitable Cas9 polypeptide includes the following substitutions: D1135L, S1136W, G1218K, E1219Q, R1335Q, and T1337R. For example, in some cases, a suitable Cas9 polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in FIG. 28A, where amino acid 1135 is Leu, amino acid 1136 is Trp, amino acid 1218 is Lys, amino acid 1219 is Gln, amino acid 1335 is Gln, and amino acid 1337 is Arg. In ... and / or T1337 (e.g., a T1337R or T1337K substitution), D1135 (e.g., a D1135L, D1135A, D1135W, or D1135F substitution), G1218 (e.g., a G1218R, G1218K, or G1218S substitution), R1335 (e.g., a R1335Q substitution), and T1337 (e.g., a T1337R or T1337K substitution).

[0093] In some cases, a suitable Cas9 polypeptide is a xCas9 polypeptide or a Cas9-NG polypeptide (see, e.g., Zhong et al. (2019) Molec. Plant 12:1027; Hu et al. (2018) Nature 556:57; and Nishimasu et al. (2018) Science 361:1259).

[0094] In some cases, the suitable CRISPR-Cas effector polypeptide is a V-type CRISPR-Cas effector polypeptide. In some cases, the V-type CRISPR-Cas effector polypeptide is a Cas12a protein. In some cases, the Cas12a protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity with any known Cas12a protein, for example, the Cas12a amino acid sequence shown in Figure 28C or Figure 28D.

[0095] In some cases, the CRISPR-Cas effector polypeptide is a CRISPR-Cas effector fusion polypeptide comprising a) a CRISPR-Cas effector polypeptide and b) one or more heterologous polypeptides (also referred to as fusion partners). In some cases, the one or more heterologous polypeptides comprise a single-stranded nuclease, a double-stranded nuclease, a helicase, a methylase, a demethylase, an acetylase, a deacetylase, a deaminase, an integrase, a recombinase, a base editor, or a prime editor. In some cases, the one or more heterologous polypeptides comprise a nuclear localization signal. In some cases, the fusion partner (heterologous polypeptide) is a reverse transcriptase. In some cases, the fusion partner is a base editor. In some cases, the fusion partner (heterologous polypeptide) is a deaminase.

[0096] In some cases, the heterologous polypeptide is a reverse transcriptase polypeptide. Thus, in some cases, the CRISPR-Cas effector polypeptide is a CRISPR-Cas effector fusion polypeptide comprising a) a CRISPR-Cas effector polypeptide and b) a reverse transcriptase. Such fusion polypeptides are useful for prime editing (e.g., Anzalone et al. (2019) Nature 576:149; and Scholefield and Harrison (2021) Gene Therapy 28:396.). In some cases, the CRISPR-Cas effector polypeptide portion of the fusion polypeptide is catalytically inactive. Suitable reverse transcriptases include, for example, murine leukemia virus reverse transcriptase, Rous sarcoma virus reverse transcriptase, human immunodeficiency virus type I reverse transcriptase, Moloney murine leukemia virus reverse transcriptase, and the like. In some cases, the fusion polypeptide comprising a CRISPR-Cas effector polypeptide and a reverse transcriptase uses a modified gRNA. For example, in some cases, the gRNA is modified to contain sequence information that is integrated into the genome near the spacer-derived CRISPR domain binding site.

[0097] In some cases, the heterologous polypeptide is a nuclease.Suitable nucleases include, but are not limited to, homing nuclease polypeptide, FokI polypeptide, transcription activator-like effector nuclease (TALEN) polypeptide, MegaTAL polypeptide, meganuclease polypeptide, zinc finger nuclease (ZFN), ARCUS nuclease, etc.Meganuclease can be engineered from LADLIDADG homing endonuclease (LHE). MegaTAL polypeptides can comprise a TALE DNA binding domain and an engineered meganuclease (see, e.g., WO2004 / 067736 (homing endonucleases); Urnov et al. (2005) Nature 435:646 (ZFNs); Mussolino et al. (2011) Nucle. Acids Res. 39:9283 (TALE nucleases); Boissel et al. (2013) Nucl. Acids Res. 42:2591 (MegaTALs)).

[0098] In some cases, the heterologous polypeptide is a base editor. Suitable base editors include, for example, adenosine deaminase, cytidine deaminase (e.g., activation-induced cytidine deaminase (AID)), APOBEC3G, and the like.

[0099] A suitable adenosine deaminase is an enzyme capable of deaminating adenosine in DNA. In some cases, the deaminase is TadA deaminase.

[0100] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 177).

[0101] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 178).

[0102] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Staphylococcus aureus TadA amino acid sequence: MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFK NLRANKKSTN: (SEQ ID NO: 179).

[0103] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Bacillus subtilis TadA amino acid sequence: MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE (SEQ ID NO: 180).

[0104] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following TadA of Salmonella typhimurium: MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV (SEQ ID NO: 181).

[0105] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Shewanella putrefaciens TadA amino acid sequence: MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE (SEQ ID NO: 182).

[0106] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following TadA amino acid sequence of Haemophilus influenzae F3031: MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTAHAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLS TFFQKRREEKKIEKALLKSLSDK (SEQ ID NO: 187).

[0107] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Caulobacter crescentus TadA amino acid sequence: MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI (SEQ ID NO: 183).

[0108] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following TadA amino acid sequence of the metal reducing bacterium Geobacter sulfurreducens: MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP (SEQ ID NO: 184).

[0109] Cytidine deaminases suitable for inclusion in a CRISPR / Cas effector polypeptide fusion polypeptide include any enzyme capable of deaminating cytidine in DNA.

[0110] In some cases, the cytidine deaminase is an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some cases, the APOBEC family deaminase is selected from the group consisting of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase. In some cases, the cytidine deaminase is an activation-induced deaminase (AID).

[0111] In some cases, a suitable cytidine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL (SEQ ID NO: 185).

[0112] In some cases, a suitable cytidine deaminase is AID and includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: MDSLLMNRRK FLYQFKNVRW AKGRRETYLC YVVKRRDSAT SFSLDFGYLR NKNGCHVELL FLRYISDWDL DPGRCYRVTW FTSWSPCYDC ARHVADFLRG NPNLSLRIFT ARLYFCEDRK AEPEGLRRLH RAGVQIAIMT FKENHERTFK AWEGLHENSV RLSRQLRRIL LPLYEVDDLR DAFRTLGL (SEQ ID NO: 186).

[0113] In some cases, a suitable cytidine deaminase is AID and includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: mdsllmnrrk flyqfknvrw akgrretylc yvvkrrdsat sfsldfgylr nkngchvell flryisdwdl dpgrcyrvtw ftswspcydc arhvadflrg npnlslrift arlyfcedrk aepeglrrlh ragvqiaimt fkdyfycwnt fvenhertfk aweglhensv rlsrqlrril lplyevddlr dafrtlgl (SEQ ID NO: 185).

[0114] In some cases, the CRISPR-Cas fusion polypeptide comprises one or more nuclear localization signals (NLSs). In some cases, the CRISPR-Cas fusion polypeptide comprises both one or more NLSs and a heterologous effector polypeptide. In some cases, the CRISPR-Cas fusion polypeptide comprises one or more NLSs (e.g., two or more, three or more, four or more, or five or more NLSs). In some cases, one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are located at or near (e.g., within 50 amino acids) the N-terminus and / or C-terminus. In some cases, one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are located at or near (e.g., within 50 amino acids) the N-terminus. In some cases, one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are located at or near (e.g., within 50 amino acids) the C-terminus. In some cases, one or more NLSs (three or more, four or more, or five or more NLSs) are located at or near (e.g., within 50 amino acids of) both the N-terminus and the C-terminus. In some cases, one NLS is located at the N-terminus and one NLS is located at the C-terminus.

[0115] Non-limiting examples of NLSs include the NLS of the large T antigen of the SV40 virus having the amino acid sequence PKKKRKV (SEQ ID NO: 188), an NLS from nuclear plasmin (e.g., the nuclear plasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 189)), a c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 190) or RQRRNELKRSP (SEQ ID NO: 191), a hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 192), a sequence of the IBB domain of importin-α RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 193), a sequence of the sarcoma T protein VSRKRPRP (SEQ ID NO: 194) and PPKKARED (SEQ ID NO: 195), a sequence of human p53 PQPKKKPL (SEQ ID NO: 196), a sequence of mouse c-abl IV sequence SALIKKKKKKMAP (SEQ ID NO: 197), influenza virus NS1 sequences DRLRR (SEQ ID NO: 198) and PKQKKRK (SEQ ID NO: 199), hepatitis virus delta antigen sequence RKLKKKIKKL (SEQ ID NO: 200), mouse Mx1 protein sequence REKKKFLKRR (SEQ ID NO: 201), human poly(ADP-ribose) polymerase sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 202), steroid hormone receptor (human) glucocorticoid sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 203), and an NLS sequence derived from the bipartite SV40 NLS with the following amino acid sequence: KRTADGSEFESPKKKRKV (SEQ ID NO: 138).

[0116] In some cases, the NLS comprises the amino acid sequence KK / RXK / R, where X is any amino acid, and the NLS is 7-17 amino acids, 5-15 amino acids, or 15-20 amino acids in length. In some cases, the NLS comprises the amino acid sequence RPAATKKAGQAKKKKLD (SEQ ID NO:204) and is 17 amino acids in length. In some cases, the NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO:188) and is 7 amino acids in length. In some cases, the NLS comprises the amino acid sequence PKKKRKVED (SEQ ID NO:205) and is 9 amino acids in length. In some cases, the NLS comprises the amino acid sequence PKKKRKVDT (SEQ ID NO:206) and is 9 amino acids in length.

[0117] In some cases, the CRISPR-Cas effector polypeptide or the CRISPR-Cas fusion polypeptide comprises a covalently linked antibody or non-antibody-based recognition scaffold. Suitable non-antibody-based recognition scaffolds include avimers, DARPins, adnectins, avimers, affibodies, anticalins, or affilins. The covalently linked antibody or non-antibody-based recognition scaffold can be linked to the CRISPR-Cas effector polypeptide via a proteolytically cleavable linker. The covalently linked antibody or non-antibody-based recognition scaffold can target the CRISPR-Cas effector polypeptide or the CRISPR-Cas fusion polypeptide to a target eukaryotic cell.

[0118] CRISPR-Cas guide nucleic acid In some cases, the cargo is a CRISPR-Cas guide nucleic acid. As mentioned above, the composition of the present disclosure comprises a) a cargo delivery fusion polypeptide of the present disclosure and b) a nucleic acid. In some cases, the nucleic acid is a CRISPR-Cas guide nucleic acid or a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas guide nucleic acid. Also, as mentioned above, in some cases, the composition of the present disclosure comprises an RNP complex comprising i) a class 2 CRISPR-Cas effector polypeptide or a nucleic acid comprising a nucleotide sequence encoding a class 2 CRISPR-Cas effector polypeptide, and ii) a guide nucleic acid or a nucleic acid comprising a nucleotide sequence encoding a guide nucleic acid.

[0119] A nucleic acid that binds to a class 2 CRISPR-Cas endonuclease (e.g., a type II, type V, or type VI CRISPR-Cas protein) and targets the complex to a specific location within a target nucleic acid is referred to herein as a guide nucleic acid (e.g., a "guide RNA" or a "CRISPR-Cas guide nucleic acid" or a "CRISPR-Cas guide RNA"). A guide nucleic acid confers target specificity to a complex (RNP complex) by including a targeting segment that includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence complementary to the sequence of a target nucleic acid.

[0120] A guide nucleic acid can be said to include two segments: a first segment (referred to herein as a "targeting segment") and a second segment (referred to herein as a "protein-binding segment"). By "segment" is meant a segment / portion / region of a molecule, e.g., a contiguous range of nucleotides in a nucleic acid molecule. A segment can also mean a region / portion of a complex, in which case a segment can include multiple molecular regions. The "targeting segment" is also referred to herein as the "variable region" of the guide RNA. The "protein-binding segment" is also referred to herein as the "constant region" of the guide RNA. In some cases, the guide RNA is a Cas9 guide RNA.

[0121] The targeting segment of the guide nucleic acid comprises a guide sequence. The "guide sequence" (also referred to as a "targeting sequence") can be modified to allow the guide RNA to target a CRISPR-Cas effector polypeptide to any desired sequence of any desired target nucleic acid, except that it can take into account the protospacer adjacent motif (PAM) sequence. The guide nucleic acid suitable for inclusion in the composition of the present disclosure comprises a targeting sequence that is complementary to a nucleotide sequence in the HBB gene that contains one or more β-thalassemia-associated mutations.

[0122] In some cases, the guide RNA is a single molecule (or "single guide") guide RNA ("sgRNA"). In some cases, the guide RNA is a double molecule (or "dual guide") guide RNA ("dgRNA").

[0123] In some cases, the total length of the guide nucleic acid (e.g., sgRNA) is between 35 nucleotides (nt) and 150 nt. In some cases, the total length of the guide nucleic acid (e.g., sgRNA) is between 35 nt and 40 nt, between 40 nt and 45 nt, between 45 nt and 50 nt, between 50 nt and 60 nt, between 60 nt and 70 nt, between 70 nt and 80 nt, between 80 nt and 90 nt, between 90 nt and 100 nt, between 100 nt and 125 nt, or between 125 nt and 150 nt.

[0124] The targeting segment of a guide nucleic acid (e.g., sgRNA) can have a length of 7 nucleotides (nt) or more (e.g., 8 nucleotides or more, 9 nucleotides or more, 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 25 nucleotides or more, 30 nucleotides or more, or 40 nucleotides or more). In some cases, the targeting segment may be 7-100 nucleotides (nt) (e.g., 7-80 nt, 7-60 nt, 7-40 nt, 7-30 nt, 7-25 nt, 7-22 nt, 7-20 nt, 7-18 nt, 8-80 nt, 8-60 nt, 8-40 nt, 8-30 nt, 8-25 nt, 8-22 nt, 8-20 nt, 8-18 nt, 10-100 nt, 10-80 nt, 10-60 nt, 10-40 nt, 10-30 nt, 10-25 nt, 10-22 nt, 10-20 nt, 10-18 nt, 12-100 nt, 12-80 nt, 12-60 nt, 12-40 nt, 12-3 The length of the nucleic acid sequence may be 0 nt, 12 to 25 nt, 12 to 22 nt, 12 to 20 nt, 12 to 18 nt, 14 to 100 nt, 14 to 80 nt, 14 to 60 nt, 14 to 40 nt, 14 to 30 nt, 14 to 25 nt, 14 to 22 nt, 14 to 20 nt, 14 to 18 nt, 16 to 100 nt, 16 to 80 nt, 16 to 60 nt, 16 to 40 nt, 16 to 30 nt, 16 to 25 nt, 16 to 22 nt, 16 to 20 nt, 16 to 18 nt, 18 to 100 nt, 18 to 80 nt, 18 to 60 nt, 18 to 40 nt, 18 to 30 nt, 18 to 25 nt, 18 to 22 nt, or 18 to 20 nt).

[0125] In some cases, a guide nucleic acid suitable for inclusion in a composition of the present disclosure comprises a nucleotide sequence that hybridizes to a contiguous stretch of about 7 nucleotides (nt) to about 50 nt (e.g., 7 nt, 8 nt, 9 nt, 10 nt, 10 nt-15 nt, 15 nt-20 nt, 20 nt-25 nt, 25 nt-30 nt, 30 nt-35 nt, 35 nt-40 nt, 40 nt-45 nt, or 45 nt-50 nt) of a target nucleic acid.

[0126] RNP In some cases, the cargo is an RNP. In some cases, the composition of the disclosure comprises: a) a cargo delivery fusion polypeptide of the disclosure; and b) an RNP comprising i) a CRISPR-Cas effector polypeptide and ii) a CRISPR-Cas guide nucleic acid. In some cases, the molar ratio of the cargo delivery fusion polypeptide to the RNP is at least 3:1. In some cases, the molar ratio of the cargo delivery fusion polypeptide to the RNP is about 3:1 to about 50:1. In some cases, the molar ratio of the cargo delivery fusion polypeptide to the RNP is about 3:1 to about 5:1, about 5:1 to about 10:1, about 10:1 to about 20:1, about 20:1 to about 30:1, about 30:1 to about 40:1, or about 40:1 to about 50:1.

[0127] Donor Nucleic Acid In some cases, the composition of the present disclosure comprises a donor nucleic acid. In some cases, the donor template nucleic acid suitable for inclusion in the composition of the present disclosure is a donor DNA template comprising a nucleotide sequence that corrects a deleterious mutation in a target nucleic acid. In some cases, the donor template nucleic acid suitable for inclusion in the composition of the present disclosure is a donor DNA template comprising a nucleotide sequence that encodes a heterologous polypeptide, such as a therapeutic polypeptide (e.g., a CAR). In some cases, the donor template is single-stranded (e.g., single-stranded DNA, ssDNA). In some cases, the donor template is double-stranded (e.g., double-stranded DNA, dsDNA). In some cases, the donor template comprises both ssDNA and dsDNA. In some cases, the donor template is present in a recombinant virus vector, such as a recombinant adeno-associated virus (AAV) vector.

[0128] "Donor nucleic acid" or "donor sequence" or "donor polynucleotide" or "donor template" or "template" or "repair template" or "homologous recombination repair template" ("HDRT") refers to a nucleic acid sequence that is inserted into a site cleaved by a CRISPR-Cas effector protein (e.g., after dsDNA cleavage, after nicking of the target DNA, after dual nicking of the target DNA, etc.). The donor polynucleotide has sufficient homology to the genomic sequence of the target site, e.g., 70%, 80%, 85%, 90%, 95%, or 100% homology to a nucleotide sequence adjacent to the target site, e.g., within about 50 bases of the target site, e.g., within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately adjacent to the target site, to aid in homologous recombination repair between the donor polynucleotide and the genomic sequence to which the donor polynucleotide has homology. Homologous recombination repair can be aided when sequence homology between the donor and genomic sequence is about 25, 50, 100, or 200 nucleotides, or greater than 200 nucleotides (or any integer value between 10 and 200 nucleotides, or greater). Donor polynucleotides can be of any length, such as 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more.

[0129] Typically, the donor sequence is not identical to the genomic sequence to be replaced. Rather, the donor sequence may contain at least one or more single base changes, insertions, deletions, inversions, or rearrangements relative to the genomic sequence, so long as there is sufficient homology to aid in homology-directed repair (e.g., gene correction, e.g., conversion of disease-causing or non-disease-causing base pairs).

[0130] In some cases, the length of the donor template DNA oligonucleotide is between 50 nucleotides and 100 nucleotides. In some cases, the length of the donor template DNA oligonucleotide is between 50 nucleotides (nt) and 60 nt, between 60 nt and 70 nt, between 70 nt and 80 nt, between 80 nt and 90 nt, or between 90 nt and 100 nt.

[0131] Methods of Delivering Cargo to Eukaryotic Cells Using the Cargo-Delivery Fusion Polypeptides of the Disclosure The present invention provides a method of delivering a cargo to a eukaryotic cell, comprising contacting the eukaryotic cell with a composition of the present invention to generate a modified eukaryotic cell comprising the cargo. The present invention provides a method of delivering a cargo to a target population of eukaryotic cells, comprising contacting the target population of eukaryotic cells with a composition of the present invention to generate a modified target population of eukaryotic cells comprising the cargo. In some cases, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more than 90% of the target cell population is modified to comprise the cargo. In some cases, the eukaryotic cell or the target population of eukaryotic cells is in vitro. In some cases, the eukaryotic cell or the target population of eukaryotic cells is in vivo.

[0132] Figure 2 shows a schematic diagram of using a cargo delivery fusion polypeptide to deliver a cargo (in this figure, the cargo is CRISPR-Cas9) to a target cell. An antibody conjugated to Cas9 binds to a cell surface receptor to trigger endocytosis, allowing internalization. The amphipathic peptide allows the internalized cargo to escape from the endosome. CRISPR-Cas9 can then translocate to the nucleus via a nuclear localization signal to perform genome editing. Although Figure 2 shows Cas9 conjugated to an antibody, in some cases, the cargo (e.g., Cas9 or other cargo) may not be conjugated to an antibody. The amphipathic peptide has cell-penetrating activity, allowing the transport of macromolecular cargo across the cell membrane without the engagement of a specific receptor.

[0133] In some cases, the method of the present disclosure is less toxic to cells than electroporation.For example, after the composition of the present disclosure is contacted with the target population of eukaryotic cells, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more than 90% of the cells of the target population continue to survive for at least 24 hours, at least 48 hours, at least 72 hours, or at least 5 days after the composition of the present disclosure is contacted with the target population of eukaryotic cells. In some cases, the methods of the disclosure provide for modification of target nucleic acids in at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the target eukaryotic cell population while maintaining a viability of at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or greater than 90% of the target cell population for a period of at least 24 hours, at least 48 hours, at least 72 hours, or at least 5 days after contacting the target cell population with a composition of the disclosure.

[0134] Eukaryotic cells that can be modified to contain cargo using the methods of the present disclosure include, for example, mammalian cells, e.g., human cells, non-human primate cells, mouse cells, etc. Mammalian cells that can be modified to contain cargo using the methods of the present disclosure include, for example, immune cells (e.g., T cells (e.g., regulatory T cells, CD4 + T cells, CD8 + These include T cells, natural killer (NK) cells, stem cells, kidney cells, and nerve cells.

[0135] Suitable cells include stem cells (e.g., embryonic stem (ES) cells, induced pluripotent stem (iPS) cells), germ cells (e.g., oocytes, sperm, oogonia, spermatogonia, etc.), somatic cells (e.g., fibroblasts, oligodendrocytes, glial cells, hematopoietic cells, neurons, muscle cells, bone cells, hepatic cells, pancreatic cells, etc.).

[0136] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone marrow derived progenitor cells, cardiomyocytes, skeletal cells, fetal cells, undifferentiated cells, multipotent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogeneic cells, allogeneic cells, and postnatal stem cells.

[0137] In some cases, the mammalian cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg).

[0138] In some cases, the mammalian cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also called somatic stem cells.

[0139] Adult stem cells reside in differentiated tissues but retain the property of self-renewal and the ability to give rise to multiple cell types, including cell types common to the tissue they are normally found in. Many examples of somatic stem cells are known to those of skill in the art, including muscle stem cells, hematopoietic stem cells, epithelial stem cells, neural stem cells, mesenchymal stem cells, mammary stem cells, intestinal stem cells, mesodermal stem cells, endothelial stem cells, olfactory stem cells, neural crest stem cells, etc.

[0140] Stem cells of interest include mammalian stem cells, where the term "mammal" refers to any animal classified as a mammal, including humans, non-human primates, farm animals, and zoo, laboratory, sports, or pet animals (dogs, horses, cats, cows, mice, rats, rabbits, etc.). In some cases, the stem cells are human stem cells. In some cases, the stem cells are rodent (mouse, rat, etc.) stem cells. In some cases, the stem cells are non-human primate stem cells.

[0141] The stem cells can express one or more stem cell markers, such as SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, and PPARGC1A.

[0142] In some cases, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesodermally derived cells that can be isolated from bone marrow, blood, umbilical cord blood, fetal liver, and yolk sac. HSCs are CD34 positive and CD38 negative (CD34 + and CD38 - ). HSCs are capable of repopulating erythroid, neutrophil-macrophage, megakaryocyte, and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some degree of self-renewal cell division and can be induced to differentiate into the same lineages as are found in vivo. Thus, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphocytes.

[0143] In other cases, the stem cell is a neural stem cell (NSC). A neural stem cell (NSC) can differentiate into neurons, glia (including oligodendrocytes, astrocytes). A neural stem cell is a multipotent stem cell that can divide many times and give rise to a daughter cell that is a neural stem cell, or a neural progenitor cell that can become a neuroblast or a glioblast (e.g., a cell that is destined to become one or more types of neurons and glial cells, respectively) under certain conditions. Methods for obtaining NSCs are well known in the art.

[0144] In other cases, the stem cells are mesenchymal stem cells (MSCs), which are originally derived from fetal mesoderm, isolated from adult bone marrow, and can differentiate to form muscle, bone, cartilage, fat, bone marrow stroma, and tendon.

[0145] In some cases, the target eukaryotic cell is a T cell (or a population of T cells, or a mixed population of cells that includes T cells). In some cases, the target eukaryotic cell is a B cell (or a population of B cells, or a mixed population of cells that includes B cells). In some cases, the target eukaryotic cell is a NK cell (or a population of NK cells, or a mixed population of cells that includes NK cells). In some cases, the target eukaryotic cell is a CD4 + T cells (or CD4 + A population of T cells, or CD4 + In some cases, the target eukaryotic cells are CD8 + T cells (or CD8 + A population of T cells, or CD8 + In some cases, the target eukaryotic cells are regulatory T cells (Tregs) (or a population of Tregs, or a mixed population of cells that includes Tregs).

[0146] In some cases, the target eukaryotic cell is an antigen-presenting cell (APC) (or a population of APCs, or a mixed population of cells that includes APCs). In some cases, the target eukaryotic cell is a dendritic cell (DC) (or a population of DCs, or a mixed population of cells that includes DCs).

[0147] In some cases, the target eukaryotic cell is a muscle cell (or a population of muscle cells, or a mixed population of cells that includes muscle cells). In some cases, the muscle cell is a skeletal muscle cell.

[0148] In some cases, the method of the present disclosure includes a) generating a modified eukaryotic cell or a target population of modified eukaryotic cells by contacting the eukaryotic cell or a target population of modified eukaryotic cells with a composition of the present disclosure, and b) introducing a second cargo into the modified eukaryotic cell or the target population of modified eukaryotic cells. In some cases, step (b) is performed using electroporation. In some cases, step (b) is performed using transfection (e.g., contacting the modified eukaryotic cell or the target population of modified eukaryotic cells with a recombinant expression vector (e.g., a recombinant viral vector) that includes a nucleotide sequence that encodes a cargo). In some cases, step (b) is performed using a second composition of the present disclosure that includes a second cargo that is different from the cargo delivered in step (a).

[0149] In some cases, the method of the present disclosure includes contacting a target eukaryotic cell or a target eukaryotic cell population with a) a composition of the present disclosure, comprising: i) a cargo delivery fusion polypeptide of the present disclosure; and ii) an RNP comprising a CRISPR-Cas effector polypeptide (or a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide) and a guide RNA (or a nucleic acid comprising a nucleotide sequence encoding a guide RNA); and b) a donor template. In some cases, the donor template is provided in a recombinant vector, such as a recombinant AAV vector. In some cases, the donor template comprises a nucleotide sequence encoding a heterologous (not naturally present in or produced by the target eukaryotic cell) polypeptide. In some cases, the donor template comprises a nucleotide sequence encoding a therapeutic polypeptide. As a non-limiting example of this embodiment, T cells obtained from a patient (e.g., a cancer patient) are contacted ex vivo with a) a composition comprising: i) a cargo delivery fusion polypeptide of the present disclosure; ii) an RNP comprising a CRISPR-Cas effector polypeptide (or a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide) and a guide RNA (or a nucleic acid comprising a nucleotide sequence encoding a guide RNA); and b) a recombinant AAV comprising a donor template encoding a chimeric antigen receptor (CAR) comprising an scFv specific for a cancer associated antigen, whereby the T cells are genetically modified to produce the CAR and express the CAR on the cell surface. The genetically modified T cells can then be introduced into the patient.

[0150] Methods for delivering cargo into eukaryotic cells The present invention provides a method for delivering cargo to a eukaryotic cell or a target population of eukaryotic cells. The method comprises contacting a eukaryotic cell with a composition comprising an amphipathic polypeptide. The cargo of interest comprises i) a DNA molecule comprising a nucleotide sequence encoding an immunogenic polypeptide, and ii) an RNP comprising a CRISPR-Cas effector polypeptide and a guide nucleic acid. In some cases, the cargo comprises a targeting moiety.

[0151] Figure 2 shows a schematic of the delivery of a cargo (in this figure the cargo is CRISPR-Cas9) to a target cell. An antibody conjugated to Cas9 binds to a cell surface receptor to trigger endocytosis, allowing internalization. The amphipathic peptide allows the internalized cargo to escape from the endosome. CRISPR-Cas9 can then translocate to the nucleus via a nuclear localization signal to perform genome editing. Although Figure 2 shows Cas9 conjugated to an antibody, in some cases the cargo (e.g., Cas9 or other cargo) may not be conjugated to an antibody. The amphipathic peptide has cell-penetrating activity, allowing the transport of macromolecular cargo across the cell membrane without the engagement of a specific receptor.

[0152] In some cases, the eukaryotic cell or target population of eukaryotic cells is in vitro. In some cases, the eukaryotic cell or target population of eukaryotic cells is in vivo. In some cases, the eukaryotic cell or target population of eukaryotic cells is ex vivo.

[0153] Eukaryotic cells that can be modified to contain cargo using the methods of the present disclosure include, for example, mammalian cells, e.g., human cells, non-human primate cells, mouse cells, etc. Mammalian cells that can be modified to contain cargo using the methods of the present disclosure include, for example, immune cells (e.g., T cells, NK cells, etc.), stem cells, kidney cells, neuronal cells, etc.

[0154] Suitable cells include stem cells (e.g., ES cells, iPS cells), germ cells (e.g., oocytes, sperm, oogonia, spermatogonia, etc.), somatic cells (e.g., fibroblasts, oligodendrocytes, glial cells, hematopoietic cells, neurons, muscle cells, bone cells, hepatic cells, pancreatic cells, etc.).

[0155] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone marrow derived progenitor cells, cardiomyocytes, skeletal cells, fetal cells, undifferentiated cells, multipotent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogeneic cells, allogeneic cells, and postnatal stem cells.

[0156] In some cases, the mammalian cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg).

[0157] In some cases, the mammalian cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also called somatic stem cells.

[0158] Adult stem cells reside in differentiated tissues but retain the property of self-renewal and the ability to give rise to multiple cell types, including cell types common to the tissue they are normally found in. Many examples of somatic stem cells are known to those of skill in the art, including muscle stem cells, hematopoietic stem cells, epithelial stem cells, neural stem cells, mesenchymal stem cells, mammary stem cells, intestinal stem cells, mesodermal stem cells, endothelial stem cells, olfactory stem cells, neural crest stem cells, etc.

[0159] Stem cells of interest include mammalian stem cells, where the term "mammal" refers to any animal classified as a mammal, including humans, non-human primates, farm animals, and zoo, laboratory, sports, or pet animals (dogs, horses, cats, cows, mice, rats, rabbits, etc.). In some cases, the stem cells are human stem cells. In some cases, the stem cells are rodent (mouse, rat, etc.) stem cells. In some cases, the stem cells are non-human primate stem cells.

[0160] The stem cells can express one or more stem cell markers, such as SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, and PPARGC1A.

[0161] In some cases, the stem cells are HSCs. In other cases, the stem cells are NSCs. In other cases, the stem cells are MSCs.

[0162] In some cases, the target eukaryotic cell is a T cell (or a population of T cells, or a mixed population of cells that includes T cells). In some cases, the target eukaryotic cell is a B cell (or a population of B cells, or a mixed population of cells that includes B cells). In some cases, the target eukaryotic cell is a NK cell (or a population of NK cells, or a mixed population of cells that includes NK cells). In some cases, the target eukaryotic cell is a CD4 + T cells (or CD4 + A population of T cells, or CD4 + In some cases, the target eukaryotic cells are CD8 + T cells (or CD8 + A population of T cells, or CD8 + In some cases, the target eukaryotic cells are regulatory T cells (Tregs) (or a population of Tregs, or a mixed population of cells that includes Tregs).

[0163] In some cases, the target eukaryotic cell is an antigen-presenting cell (APC) (or a population of APCs, or a mixed population of cells that includes APCs). In some cases, the target eukaryotic cell is a dendritic cell (DC) (or a population of DCs, or a mixed population of cells that includes DCs).

[0164] In some cases, the RNP delivered to the cell comprises a CRISPR-Cas effector polypeptide or a CRISPR-Cas fusion polypeptide comprising a covalently linked antibody or non-antibody-based recognition scaffold. Suitable non-antibody-based recognition scaffolds include avimers, DARPins, adnectins, avimers, affibodies, anticalins, or affilins. The covalently linked antibody or non-antibody-based recognition scaffold can be linked to the CRISPR-Cas effector polypeptide via a proteolytically cleavable linker. The covalently linked antibody or non-antibody-based recognition scaffold can target the CRISPR-Cas effector polypeptide or CRISPR-Cas fusion polypeptide to the target eukaryotic cell.

[0165] Amphipathic polypeptides and compositions Amphipathic polypeptides suitable for use in the methods of the present disclosure include i) endosomolytic polypeptides, and ii) cell-penetrating polypeptides.

[0166] Suitable endosomolytic polypeptides include, for example, polypeptides comprising the amino acid sequence: GLFEAIAEFIENGWEGLIEGWYG (SEQ ID NO: 163), or polypeptides having 1 to 5 amino acid substitutions relative to GLFEAIAEFIENGWEGLIEGWYG (SEQ ID NO: 163), and the length of the endosomolytic polypeptide is about 20 to about 25 amino acids.

[0167] Suitable endosomolytic polypeptides include, for example, polypeptides comprising the amino acid sequence: GLFEAIEGFIENGWEGMIDGWYG (SEQ ID NO: 164), or polypeptides having 1 to 5 amino acid substitutions relative to GLFEAIEGFIENGWEGMIDGWYG (SEQ ID NO: 164), and the length of the endosomolytic polypeptide is about 20 to about 25 amino acids.

[0168] Suitable cell-permeable polypeptides include, for example, YGRKKRRQRRR (SEQ ID NO: 207), YGRKKRRQRR (SEQ ID NO: 160), or GRKKRRQRRR (SEQ ID NO: 161), and the length of the cell-permeable polypeptide is 10 to 15 amino acids.

[0169] In some cases, the total length of the amphipathic polypeptide is between 35 amino acids and 50 amino acids. In some cases, the total length of the amphipathic polypeptide is between 35 amino acids and 40 amino acids. In some cases, the total length of the amphipathic polypeptide is between 35 amino acids and 45 amino acids. In some cases, the total length of the amphipathic polypeptide is between 40 amino acids and 45 amino acids. In some cases, the total length of the amphipathic polypeptide is between 40 amino acids and 50 amino acids.

[0170] In some cases, a suitable amphipathic polypeptide comprises an amino acid sequence of any one of the peptides designated as peptide numbers 1 to 60 in FIG. 1. In some cases, a suitable amphipathic polypeptide comprises an amino acid sequence having 1 to 5 amino acid substitutions relative to any one of the peptides designated as peptide numbers 1 to 60 in FIG. 1. In some cases, the total length of the amphipathic polypeptide is 35 to 50 amino acids. In some cases, the total length of the amphipathic polypeptide is 35 to 40 amino acids. In some cases, the total length of the amphipathic polypeptide is 35 to 45 amino acids. In some cases, the total length of the amphipathic polypeptide is 40 to 45 amino acids. In some cases, the total length of the amphipathic polypeptide is 40 to 50 amino acids.

[0171] In some cases, a suitable amphipathic polypeptide comprises an amino acid sequence of any one of the peptides designated as peptide numbers 1-18 or peptide numbers 20-37 in FIG. 1. In some cases, a suitable amphipathic polypeptide comprises an amino acid sequence having 1-5 amino acid substitutions relative to any one of the peptides designated as peptide numbers 1-18 or peptide numbers 20-37 in FIG. 1. In some cases, the total length of the amphipathic polypeptide is 35 amino acids to 50 amino acids. In some cases, the total length of the amphipathic polypeptide is 35 amino acids to 40 amino acids. In some cases, the total length of the amphipathic polypeptide is 35 amino acids to 45 amino acids. In some cases, the total length of the amphipathic polypeptide is 40 amino acids to 45 amino acids. In some cases, the total length of the amphipathic polypeptide is 40 amino acids to 50 amino acids.

[0172] In some cases, a suitable amphipathic polypeptide comprises an amino acid sequence of any one of the peptides designated in FIG. 1 as peptide number 19 or peptide numbers 40-60. In some cases, a suitable amphipathic polypeptide comprises an amino acid sequence having 1-5 amino acid substitutions relative to any one of the peptides designated in FIG. 1 as peptide number 19 or peptide numbers 40-60. In some cases, the total length of the amphipathic polypeptide is 35 amino acids to 50 amino acids. In some cases, the total length of the amphipathic polypeptide is 35 amino acids to 40 amino acids. In some cases, the total length of the amphipathic polypeptide is 35 amino acids to 45 amino acids. In some cases, the total length of the amphipathic polypeptide is 40 amino acids to 45 amino acids. In some cases, the total length of the amphipathic polypeptide is 40 amino acids to 50 amino acids.

[0173] In some cases, the amphipathic polypeptide composition of the present disclosure comprises a) an amphipathic polypeptide, and b) DMSO. In some cases, the amphipathic polypeptide is maintained in a solution containing DMSO at a concentration of about 9% DMSO to about 15% DMSO (e.g., about 10% DMSO) for a period of time before being contacted with a cargo. If the amphipathic polypeptide is prepared in a DMSO solution of less than 10%, the peptides may bind to each other and may not effectively bind to the cargo. Thus, in some cases, after the amphipathic polypeptide is held in a solution of about 10% DMSO for a period of time, the peptide is contacted with a cargo present in a solution that does not contain DMSO (e.g., a buffered aqueous solution). Thus, the amphipathic polypeptide / cargo solution may comprise 1% DMSO to 5% DMSO.

[0174] In some cases, the composition of the present disclosure comprises a) an amphipathic polypeptide, and b) saline (e.g., 0.9% NaCl). In some cases, the composition is sterile. In some cases, the pharmaceutical composition of the present invention is suitable for administration to a human subject, for example, when the composition is sterile and free of detectable pyrogens and / or other toxins. Thus, the present disclosure provides a composition comprising a) an amphipathic polypeptide, and b) saline (e.g., 0.9% NaCl), the composition being sterile and free of detectable pyrogens and / or other toxins. In some cases, the composition further comprises a cargo to be delivered.

[0175] cargo As noted above, cargoes of interest include i) DNA molecules comprising a nucleotide sequence encoding an immunogenic polypeptide, and ii) RNPs comprising a CRISPR-Cas effector polypeptide and a guide nucleic acid.

[0176] DNA encoding an immunogenic polypeptide In some cases, the cargo is a DNA molecule comprising a nucleotide sequence encoding an immunogenic polypeptide. The immunogenic protein is suitable for stimulating an immune response against an antigenic protein in a mammalian host (e.g., human, non-human primate, bovine (e.g., cow), ovine (e.g., sheep), equine (e.g., horse), porcine (e.g., pig), etc.). The immunogenic polypeptide may be derived from a self-antigen, an allergen, a tumor-associated antigen, a pathogenic virus, a pathogenic bacterium, a pathogenic protozoan, a pathogenic helminth, or other pathogenic organism that infects a mammalian host. A suitable immunogenic polypeptide may be derived from any of a variety of pathogens as described above.

[0177] RNP In some cases, the cargo is an RNP comprising a CRISPR-Cas effector polypeptide and a guide nucleic acid. Suitable CRISPR-Cas effector polypeptides and guide nucleic acids are described above. In some cases, the molar ratio of the cargo delivery fusion polypeptide to the RNP is at least 3:1. In some cases, the molar ratio of the cargo delivery fusion polypeptide to the RNP is about 3:1 to about 50:1. In some cases, the molar ratio of the cargo delivery fusion polypeptide to the RNP is about 3:1 to about 5:1, about 5:1 to about 10:1, about 10:1 to about 20:1, about 20:1 to about 30:1, about 30:1 to about 40:1, or about 40:1 to about 50:1.

[0178] In some cases, the method of the disclosure includes contacting a target eukaryotic cell, or a target eukaryotic cell population, with a) a composition of the disclosure, the composition comprising: (i) an amphipathic polypeptide (as described above); ii) an RNP comprising a CRISPR-Cas effector polypeptide (or a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide) and a guide RNA (or a nucleic acid comprising a nucleotide sequence encoding a guide RNA); and b) a donor template. In some cases, the donor template is provided in a recombinant vector, such as a recombinant AAV vector. As a non-limiting example of this embodiment, T cells obtained from a patient (e.g., a cancer patient) are contacted ex vivo with a) a composition comprising: i) an amphipathic polypeptide (as described above); ii) an RNP comprising a CRISPR-Cas effector polypeptide (or a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide) and a guide RNA (or a nucleic acid comprising a nucleotide sequence encoding a guide RNA); and b) a recombinant AAV comprising a donor template encoding a chimeric antigen receptor (CAR) comprising an scFv specific for a cancer-associated antigen, whereby the T cells are genetically modified to produce the CAR and express the CAR on the cell surface. The genetically modified T cells can then be introduced into the patient.

[0179] In some cases, the RNP delivered to the cell comprises a CRISPR-Cas effector polypeptide or a CRISPR-Cas fusion polypeptide comprising a covalently linked antibody or non-antibody-based recognition scaffold. Suitable non-antibody-based recognition scaffolds include avimers, DARPins, adnectins, avimers, affibodies, anticalins, or affilins. The covalently linked antibody or non-antibody-based recognition scaffold can be linked to the CRISPR-Cas effector polypeptide via a proteolytically cleavable linker. The covalently linked antibody or non-antibody-based recognition scaffold can target the CRISPR-Cas effector polypeptide or CRISPR-Cas fusion polypeptide to the target eukaryotic cell.

[0180] composition The cargo delivered using the method of the present disclosure can be present in a composition. For example, the composition can include, in addition to the amphipathic polypeptide and the cargo, one or more of the following: salts, such as NaCl, MgCl2, KCl, MgSO4, etc.; buffers, such as 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.; solubilizers; detergents, such as non-ionic detergents, such as Tween-20; nuclease inhibitors, etc.

[0181] Such compositions can be administered to an individual in need thereof using any of a variety of routes of administration, including local and systemic routes of administration. Suitable routes of administration include intravenous, intramuscular, subcutaneous, peritumoral, etc. In some cases, the composition can be administered to an individual in need thereof by administering it into or near a target organ.

[0182] Examples of Non-Limiting Aspects of the Disclosure Mode Set A The above aspects, including the embodiments of the subject matter of the present invention, may be useful alone or in combination with one or more other aspects or embodiments.Without limiting the above description, certain non-limiting aspects of the present disclosure are presented below.As will be clear to those skilled in the art upon reading this disclosure, each of the individually numbered aspects can be used or combined with any of the preceding or succeeding individually numbered aspects.This is intended to give support for all such combinations of each aspect, and is not limited to the combination of aspects explicitly shown below.

[0183] Aspect 1. a) an endosomolytic polypeptide; b) a cell-penetrating polypeptide; A cargo delivery fusion polypeptide comprising: An amino acid sequence of a formula selected from: i) KLFEX1IEGFIENGWEX2MIDX3WX4GX5GRKKRRQRR (SEQ ID NO: 165), X1 is A, R, or K; X2 is A or G; X3 is L or G; X4 is N or Y; X5, if present, is Y; ii) X1LFEX2IEGFIENGWEGMIDGWYGYGRKKRRQRR (SEQ ID NO: 166), X1 is R or G; X2 is R or K; iii) GLFEAIEGFIENGWEX1MIDX2WNGYGRKKRRQRR (SEQ ID NO: 167), X1 is A or G; X2 is G or L; iv) GLFEAIEGFIENGWEX1X2IX3LWYGYGRKKRRQRR (SEQ ID NO: 168), X1 is A or G; X2 is L or M; X3 is D or E, and v) GLFX1AIAX2FIX3NGWX4GLIX5GWYGGRKKRRQRRR (SEQ ID NO: 208), wherein each of X1, X2, X3, X4, and X5 is independently a non-coded amino acid. Including, The cargo delivery fusion polypeptide has a length of about 32 amino acids to about 35 amino acids.

[0184] Embodiment 2. The polypeptide comprises an amino acid sequence of the formula: KLFEX1IEGFIENGWEX2MIDX3WX4GX5GRKKRRQRR (SEQ ID NO: 165), wherein: X1 is A, R, or K; X2 is A or G; X3 is L or G; X4 is N or Y; The fusion polypeptide of embodiment 1, wherein X5, if present, is Y.

[0185] Aspect 3. KLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 19, SEQ ID NO: 19), KLFEAIEGFIENGWEGMIDGWYG GRKKRRQRR (Peptide 40, SEQ ID NO: 40), KLFERIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 44, SEQ ID NO: 44), KLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 45, SEQ ID NO: 45), KLFEAIEGFIENGWEAMIDGWYGYGRKKRRQRR (Peptide 46, SEQ ID NO: 46), KLFEAIEGFIENGWEGMIDLWYGYGRKKRRQRR (Peptide 47, SEQ ID NO: 47), KLFEAIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 48, SEQ ID NO: 48), KLFEAIEGFIENGWEAMIDLWYGYGRKKRRQRR (Peptide 53, SEQ ID NO:53), KLFEAIEGFIENGWEAMIDGWNGYGRKKRRQRR (Peptide 54, SEQ ID NO:54), KLFEAIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 55, SEQ ID NO:55), KLFEAIEGFIENGWEAMIDLWNGYGRKKRRQRR (Peptide 56, SEQ ID NO:56), and KLFEKIEGFIENGWEAMIDLWNGYGRKKRRQRR (Peptide 57, SEQ ID NO:57) The fusion polypeptide of embodiment 2, comprising an amino acid sequence selected from the group consisting of:

[0186] Embodiment 4. The fusion polypeptide of embodiment 1, comprising an amino acid sequence of the formula: X1LFEX2IEGFIENGWEGMIDGWYGYGRKKRRQRR (SEQ ID NO: 166), wherein X1 is R or G and X2 is R or K.

[0187] Aspect 5. RLFERIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 42, SEQ ID NO: 42); RLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 43, SEQ ID NO: 43), and GLFERIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 41, SEQ ID NO: 41) The fusion polypeptide of embodiment 4, comprising an amino acid sequence selected from the group consisting of:

[0188] Embodiment 6. The fusion polypeptide of embodiment 1, comprising an amino acid sequence of the formula: GLFEAIEGFIENGWEX1MIDX2WNGYGRKKRRQRR (SEQ ID NO: 167), wherein X1 is A or G and X2 is G or L.

[0189] Aspect 7. GLFEAIEGFIENGWEAMIDGWNGYGRKKRRQRR (Peptide 50, SEQ ID NO:50), GLFEAIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 51, SEQ ID NO:51), and GLFEAIEGFIENGWEAMIDLWNGYGRKKRRQRR (Peptide 51, SEQ ID NO:52) The fusion polypeptide of embodiment 6, comprising an amino acid sequence selected from the group consisting of:

[0190] Embodiment 8. The peptide comprises an amino acid sequence of the formula: GLFEAIEGFIENGWEX1X2IX3LWYGYGRKKRRQRR (SEQ ID NO: 168), wherein: X1 is A or G; X2 is L or M; The fusion polypeptide of embodiment 1, wherein X3 is D or E.

[0191] 9. Amino acid sequence: GLFEAIEGFIENGWEAMIDLWYGYGRKKRRQRR (Peptide 49, SEQ ID NO: 49), or GLFEAIEGFIENGWEGLIELWYGYGRKKRRQRR (Peptide 58, SEQ ID NO:58) The fusion polypeptide of embodiment 8, comprising:

[0192] Embodiment 10. The fusion polypeptide of embodiment 1, comprising an amino acid sequence of the formula: GLFX1AIAX2FIX3NGWX4GLIX5GWYGGRKKRRQRRR (SEQ ID NO: 208), wherein each of Xi, X2, X3, X4, and X5 is independently a non-coded amino acid.

[0193] 11. Amino acid sequence: GLFαAIAαFIαNGWαGLIαGWYGGRKKRRQRRR (peptide 59, SEQ ID NO:59), or GLFαAIAαFIENGWEGLIDGWYGGRKKRRQRRR (Peptide 60, SEQ ID NO: 60) 11. The fusion polypeptide of embodiment 10, comprising:

[0194] Embodiment 12. The fusion polypeptide of embodiment 10 or embodiment 11, wherein each of X1, X2, X3, X4, and X5 is α-aminoadipic acid.

[0195] Embodiment 13. A composition comprising the cargo delivery fusion polypeptide of any one of embodiments 1 to 12.

[0196] Embodiment 14 The composition of embodiment 13, further comprising a cargo, said cargo comprising one or more of a nucleic acid, a polypeptide, and a ribonucleoprotein complex.

[0197] Embodiment 15 The composition of embodiment 14, wherein said cargo comprises a targeting moiety.

[0198] Embodiment 16 The composition of embodiment 13, comprising a nucleic acid comprising a nucleotide sequence encoding a gene product of interest.

[0199] Embodiment 17 The composition of embodiment 16, wherein said gene product of interest is an antigen.

[0200] Embodiment 18 The composition of embodiment 16 or embodiment 7, wherein said nucleic acid is a recombinant expression vector.

[0201] Embodiment 19 The composition of embodiment 18, wherein said recombinant expression vector is a recombinant viral vector.

[0202] Aspect 20. a) a CRISPR-Cas effector polypeptide, or b) a fusion polypeptide, i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides; The fusion polypeptide comprising 14. The composition of embodiment 13, comprising:

[0203] Embodiment 21 The composition of embodiment 20, comprising a CRISPR-Cas guide nucleic acid.

[0204] Embodiment 22. The composition of embodiment 21, comprising a donor DNA template.

[0205] Embodiment 23 The composition of any one of embodiments 20 to 22, wherein said CRISPR-Cas effector polypeptide is a Type II CRISPR-Cas effector polypeptide, a Type V CRISPR-Cas effector polypeptide, or a Type VI CRISPR-Cas effector polypeptide.

[0206] Embodiment 24 The composition of embodiment 21, wherein the CRISPR-Cas guide nucleic acid is RNA.

[0207] Embodiment 25 The composition of embodiment 24, wherein said CRISPR-Cas guide nucleic acid is a single-molecule guide RNA or a bi-molecule guide RNA.

[0208] Embodiment 26. A CRISPR-Cas effector fusion polypeptide comprising i) a CRISPR-Cas effector fusion polypeptide and ii) one or more nuclear localization signals. 26. The composition of any one of embodiments 20-25, comprising:

[0209] Embodiment 27. A CRISPR-Cas effector fusion polypeptide comprising i) a CRISPR-Cas effector polypeptide and ii) one or more heterologous effector polypeptides. 26. The composition of any one of embodiments 20-25, comprising:

[0210] Embodiment 28. The composition of embodiment 27, wherein at least one of said one or more heterologous effector polypeptides is a single-stranded nuclease, double-stranded nuclease, helicase, methylase, demethylase, acetylase, deacetylase, deaminase, integrase, recombinase, base editor, or prime editor.

[0211] Embodiment 29 The composition of any one of embodiments 20 to 28, wherein said CRISPR-Cas effector polypeptide or said CRISPR-Cas effector fusion polypeptide comprises a covalently linked targeting moiety.

[0212] Embodiment 30 The composition of embodiment 29, wherein said targeting moiety is Protein A, Protein G, an aptamer, a DARPin, or an antibody.

[0213] Embodiment 31 The composition of embodiment 30, comprising an antibody non-covalently bound to an affinity moiety.

[0214] Embodiment 32 The composition of embodiment 30 or embodiment 31, wherein said antibody specifically binds to an epitope on the surface of a eukaryotic cell, thereby targeting said composition to said cell.

[0215] Embodiment 33 The composition of any one of embodiments 20 to 32, wherein said CRISPR-Cas effector polypeptide or said CRISPR-Cas effector fusion polypeptide comprises a non-polypeptide polymer.

[0216] Embodiment 34 The composition of embodiment 33, wherein the non-polypeptide polymer is poly(ethylene glycol).

[0217] Embodiment 35. The composition of any one of embodiments 21 to 34, wherein said CRISPR-Cas effector polypeptide and said guide nucleic acid are present in a ribonucleoprotein (RNP) complex.

[0218] Embodiment 36 The composition of embodiment 35, wherein the molar ratio of cargo delivery fusion polypeptide to RNP is at least 3:1.

[0219] Embodiment 37. The composition of embodiment 36, wherein the molar ratio of cargo delivery fusion polypeptide to RNP is between 10:1 and 50:1.

[0220] Embodiment 38 The composition of any one of embodiments 13 to 37, wherein said cargo delivery fusion polypeptide is present in the composition at a concentration of from about 2 μM to about 50 μM.

[0221] Embodiment 39. The composition of any one of embodiments 13 to 38, further comprising one or more of a solubilizing agent, a surfactant, a buffer, a salt, and a protease inhibitor.

[0222] Embodiment 40. The composition of any one of embodiments 13 to 38, comprising poly(ethylene glycol), a non-ionic surfactant, or both.

[0223] Embodiment 41. A method for delivering a cargo to a target population of eukaryotic cells, comprising contacting said cells with a composition of any one of embodiments 14 to 38, thereby generating a modified target population of eukaryotic cells comprising said cargo.

[0224] Embodiment 42 The method of embodiment 41, wherein said target population of eukaryotic cells comprises T cells, stem cells, natural killer cells, renal cells, or neuronal cells.

[0225] Embodiment 43 The method of embodiment 41, wherein said target eukaryotic cell is a hematopoietic stem cell or hematopoietic progenitor cell.

[0226] Embodiment 44 The method of any one of embodiments 41 to 43, wherein the cell is in vitro.

[0227] Embodiment 45 The method of embodiment 44, wherein after said contacting step, at least 50% of said target population of eukaryotic cells retain viability.

[0228] Embodiment 46 The method of embodiment 44 or embodiment 45, comprising a step of introducing a second composition comprising a second cargo into said modified target population of eukaryotic cells.

[0229] Embodiment 47 The method of embodiment 46, wherein said introducing step is by electroporation or transfection.

[0230] Embodiment 48 The method of embodiment 47, wherein said transfection comprises contacting said modified target population of eukaryotic cells with a recombinant viral vector.

[0231] Embodiment 49 The method of any one of embodiments 41 to 43, wherein the cell is in vivo.

[0232] 50. A method for delivering a DNA molecule to a eukaryotic cell, comprising: a) an amphipathic cargo-delivery fusion polypeptide, i) an endosomolytic polypeptide; and ii) a cell-penetrating polypeptide; and b) a DNA molecule comprising a nucleotide sequence encoding an immunogenic polypeptide. A composition comprising The method comprises the step of contacting the

[0233] Embodiment 51 The method of embodiment 50, wherein said immunogenic polypeptide is a viral polypeptide.

[0234] 52. The endosomolytic polypeptide comprises: a) contains the amino acid sequence GLFEAIAEFIENGWEGLIEGWYG (SEQ ID NO: 163), b) containing 1 to 5 amino acid substitutions relative to GLFEAIAEFIENGWEGLIEGWYG (SEQ ID NO: 163), or c) comprises the amino acid sequence GLFEAIEGFIENGWEGMIDGWYG (SEQ ID NO: 164); or d) GLFEAIEGFIENGWEGMIDGWYG (SEQ ID NO: 164) containing 1 to 5 amino acid substitutions; 52. The method of embodiment 50 or embodiment 51, wherein said endosomolytic polypeptide has a length of about 20 amino acids to about 30 amino acids.

[0235] Embodiment 53. The method of any one of embodiments 50 to 52, wherein the cell-permeable polypeptide comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 207), YGRKKRRQRR (SEQ ID NO: 160), or GRKKRRQRRR (SEQ ID NO: 161), and has a length of 10 amino acids to 15 amino acids.

[0236] 54. A method for delivering a ribonucleoprotein (RNP) to a eukaryotic cell, comprising: a) an amphipathic cargo-delivery fusion polypeptide, i) an endosomolytic polypeptide; and ii) a cell-penetrating polypeptide; and b) an RNP, i) a CRISPR-Cas effector polypeptide; and ii) a guide nucleic acid; The RNP A composition comprising The method comprises the step of contacting the

[0237] 55. The endosomolytic polypeptide comprises: a) contains the amino acid sequence GLFEAIAEFIENGWEGLIEGWYG (SEQ ID NO: 163), b) containing 1 to 5 amino acid substitutions relative to GLFEAIAEFIENGWEGLIEGWYG (SEQ ID NO: 163), or c) comprises the amino acid sequence GLFEAIEGFIENGWEGMIDGWYG (SEQ ID NO: 164); or d) GLFEAIEGFIENGWEGMIDGWYG (SEQ ID NO: 164) containing 1 to 5 amino acid substitutions; 55. The method of embodiment 54, wherein the endosomolytic polypeptide has a length of about 20 amino acids to about 30 amino acids.

[0238] Embodiment 56. The method of embodiment 54 or embodiment 55, wherein the cell-permeable polypeptide comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 207), YGRKKRRQRR (SEQ ID NO: 160), or GRKKRRQRRR (SEQ ID NO: 161), and has a length of 10 amino acids to 15 amino acids.

[0239] Embodiment 57 The method of any one of embodiments 54 to 56, comprising the step of introducing a DNA donor template into said cell.

[0240] Embodiment 58 The method of embodiment 57, wherein said donor template is present in a recombinant viral vector.

[0241] Embodiment 59 The method of embodiment 58, wherein said recombinant viral vector is a recombinant adeno-associated viral vector.

[0242] Embodiment 60 The method of any one of embodiments 57 to 59, wherein the donor template comprises a nucleotide sequence encoding a polypeptide.

[0243] Embodiment 61 The method of embodiment 60, wherein the polypeptide is a chimeric antigen receptor comprising a single chain Fv or a nanobody specific for a cancer-associated antigen.

[0244] Embodiment 62 The method of any one of embodiments 54 to 61, wherein the eukaryotic cell is an immune cell.

[0245] Embodiment 63 The method of embodiment 62, wherein the immune cell is a T cell.

[0246] Embodiment 64 The method of any one of embodiments 54 to 63, wherein the eukaryotic cell is in vivo.

[0247] Embodiment 65 The method of any one of embodiments 54 to 63, wherein the eukaryotic cell is in vitro.

[0248] Mode Set B The above aspects, including the embodiments of the subject matter of the present invention, may be useful alone or in combination with one or more other aspects or embodiments.Without limiting the above description, certain non-limiting aspects of the present disclosure are presented below.As will be clear to those skilled in the art upon reading this disclosure, each of the individually numbered aspects can be used or combined with any of the preceding or succeeding individually numbered aspects.This is intended to give support for all such combinations of each aspect, and is not limited to the combination of aspects explicitly shown below.

[0249] Aspect 1. A cargo delivery fusion polypeptide comprising: a) an endosomolytic polypeptide; and b) a cell-penetrating polypeptide, the cargo delivery fusion polypeptide comprising any one of the amino acid sequences of formulas I to VIII, having a length of about 32 amino acids to about 35 amino acids, and wherein any two adjacent amino acids are independently linked by an amide bond or a non-amide bond.

[0250] Embodiment 2. Formula I: KLFEX1IEGFIENGWEX2MIDX3WX4GX5GRKKRRQRR (SEQ ID NO: 165), comprising the amino acid sequence X1 is A, R, or K; X2 is A or G; X3 is L or G; X4 is N or Y; The fusion polypeptide of embodiment 1, wherein X5, if present, is Y.

[0251] Aspect 3. KLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 19, SEQ ID NO: 19), KLFEAIEGFIENGWEGMIDGWYG GRKKRRQRR (Peptide 40, SEQ ID NO: 40), KLFERIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 44, SEQ ID NO: 44), KLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 45, SEQ ID NO: 45), KLFEAIEGFIENGWEAMIDGWYGYGRKKRRQRR (Peptide 46, SEQ ID NO: 46), KLFEAIEGFIENGWEGMIDLWYGYGRKKRRQRR (Peptide 47, SEQ ID NO: 47), KLFEAIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 48, SEQ ID NO: 48), KLFEAIEGFIENGWEAMIDLWYGYGRKKRRQRR (Peptide 53, SEQ ID NO:53), KLFEAIEGFIENGWEAMIDGWNGYGRKKRRQRR (Peptide 54, SEQ ID NO:54), KLFEAIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 55, SEQ ID NO:55), KLFEAIEGFIENGWEAMIDLWNGYGRKKRRQRR (Peptide 56, SEQ ID NO:56), and KLFEKIEGFIENGWEAMIDLWNGYGRKKRRQRR (Peptide 57, SEQ ID NO:57) The fusion polypeptide of embodiment 2, comprising an amino acid sequence selected from the group consisting of:

[0252] Embodiment 4. The fusion polypeptide of embodiment 1, comprising an amino acid sequence of formula II: X1LFEX2IEGFIENGWEGMIDGWYGYGRKKRRQRR (SEQ ID NO: 166), wherein X1 is R or G and X2 is R or K.

[0253] Aspect 5. RLFERIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 42, SEQ ID NO: 42); RLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 43, SEQ ID NO: 43), and GLFERIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 41, SEQ ID NO: 41) The fusion polypeptide of embodiment 4, comprising an amino acid sequence selected from the group consisting of:

[0254] Embodiment 6. The fusion polypeptide of embodiment 1, comprising an amino acid sequence of formula III: GLFEAIEGFIENGWEX1MIDX2WNGYGRKKRRQRR (SEQ ID NO: 167), wherein X1 is A or G and X2 is G or L.

[0255] Aspect 7. GLFEAIEGFIENGWEAMIDGWNGYGRKKRRQRR (Peptide 50, SEQ ID NO:50), GLFEAIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 51, SEQ ID NO:51), and GLFEAIEGFIENGWEAMIDLWNGYGRKKRRQRR (Peptide 51, SEQ ID NO:51) The fusion polypeptide of embodiment 6, comprising an amino acid sequence selected from the group consisting of:

[0256] Embodiment 8. Formula IV: GLFEAIEGFIENGWEX1X2IX3LWYGYGRKKRRQRR (SEQ ID NO: 168), comprising the amino acid sequence X1 is A or G; X2 is L or M; The fusion polypeptide of embodiment 1, wherein X3 is D or E.

[0257] 9. Amino acid sequence: GLFEAIEGFIENGWEAMIDLWYGYGRKKRRQRR (Peptide 49, SEQ ID NO: 49), or GLFEAIEGFIENGWEGLIELWYGYGRKKRRQRR (Peptide 58, SEQ ID NO:58) The fusion polypeptide of embodiment 8, comprising:

[0258] Embodiment 10. The fusion polypeptide of embodiment 1, comprising an amino acid sequence of formula V: GLFX1AIAX2FIX3NGWX4GLIX5GWYGGRKKRRQRRR (SEQ ID NO: 208), wherein each of Xi, X2, X3, X4, and X5 is independently a non-coded amino acid.

[0259] 11. Amino acid sequence: GLFαAIAαFIαNGWαGLIαGWYGGRKKRRQRRR (peptide 59, SEQ ID NO:59), or GLFαAIAαFIENGWEGLIDGWYGGRKKRRQRRR (Peptide 60, SEQ ID NO: 60) 11. The fusion polypeptide of embodiment 10, comprising:

[0260] Aspect 12. X1, X2, X 3、 The fusion polypeptide of embodiment 10 or embodiment 11, wherein each of X4 and X5 is α-aminoadipic acid.

[0261] Embodiment 13. The fusion polypeptide of embodiment 1, comprising an amino acid sequence of formula VI: KLFEX1IX2X3FIENGWEGMIX4X5WX6GYGRKKRRQRX7 (SEQ ID NO: 170), wherein Xi is A or H, X2 is E or A, X3 is G or E, X4 is D or E, X5 is G or L, X6 is E, H, K, R, or N, and X7, if present, is R.

[0262] Aspect 14. KLFEAIEGFIENGWEGMIDLWEGYGRKKRRQRR (Peptide 62, SEQ ID NO: 62), KLFEAIEGFIENGWEGMIDLWHGYGRKKRRQRR (Peptide 63, SEQ ID NO: 63), KLFEAIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 64, SEQ ID NO: 64), KLFEAIEGFIENGWEGMIDLWRGYGRKKRRQRR (Peptide 65, SEQ ID NO: 65), KLFEAIEGFIENGWEGMIDLWNGYGRKKRRQR (Peptide 69, SEQ ID NO: 69), KLFEAIEGFIENGWEGMIELWNGYGRKKRRQRR (Peptide 71, SEQ ID NO: 71), KLFEAIAEFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 72, SEQ ID NO: 72), KLFEHIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 105, SEQ ID NO: 105), KLFEHIEGFIENGWEGMIDLWYGYGRKKRRQRR (peptide 107, SEQ ID NO: 107), and KLFEHIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 109, SEQ ID NO: 109) 14. The fusion polypeptide of embodiment 13, comprising an amino acid sequence selected from:

[0263] Embodiment 15. The fusion polypeptide of embodiment 1, comprising an amino acid sequence of formula VII: GLFEX1IX2X3FIENGWEGMIDX4WX5GYGRKKRRQRR (SEQ ID NO: 171), wherein Xi is R, H, A, or K, X2 is E or A, X3 is G or E, X4 is L or G, and X5 is N, Y, K, or E.

[0264] Aspect 16. GLFERIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 66, SEQ ID NO: 66); GLFEHIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 68, SEQ ID NO: 68); GLFEAIAEFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 70, SEQ ID NO: 70), GLFEKIEGFIENGWEAMIDGWYGYGRKKRRQRR (Peptide 73, SEQ ID NO: 73), GLFEKIEGFIENGWEGMIDLWYGYGRKKRRQRR (Peptide 74, SEQ ID NO: 74); GLFEAIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 75, SEQ ID NO: 31), GLFEAIEEFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 76, SEQ ID NO: 24), GLFEKIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 98, SEQ ID NO: 98); GLFEKIEGFIENGWEGMIDGWKGYGRKKRRQRR (Peptide 99, SEQ ID NO: 99); GLFEKIEGFIENGWEGMIDGWEGYGRKKRRQRR (Peptide 100, SEQ ID NO: 100), GLFEKIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 101, SEQ ID NO: 101), GLFEKIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 102, SEQ ID NO: 102), and GLFEKIEGFIENGWEGMIDLWEGYGRKKRRQRR (Peptide 103, SEQ ID NO: 103) 16. The fusion polypeptide of embodiment 15, comprising an amino acid sequence selected from:

[0265] Embodiment 17. The fusion polypeptide of embodiment 1, comprising an amino acid sequence of formula VIII: HLFEX1IEGFIENGWEGMIDX2WX3GYGRKKRRQRR (SEQ ID NO: 172), wherein Xi is A or K, X2 is G or L, and X3 is N, K, E, or Y.

[0266] Aspect 18. HLFEAIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 92, SEQ ID NO: 92), HLFEAIEGFIENGWEGMIDGWKGYGRKKRRQRR (Peptide 93, SEQ ID NO: 93), HLFEAIEGFIENGWEGMIDGWEGYGRKKRRQRR (Peptide 94, SEQ ID NO: 94), HLFEAIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 95, SEQ ID NO: 67), HLFEAIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 96, SEQ ID NO: 96), HLFEAIEGFIENGWEGMIDLWEGYGRKKRRQRR (Peptide 97, SEQ ID NO: 97), HLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 104, SEQ ID NO: 104), HLFEKIEGFIENGWEGMIDLWYGYGRKKRRQRR (Peptide 106, SEQ ID NO: 106), and HLFEKIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 108, SEQ ID NO: 108) 18. The fusion polypeptide of embodiment 17, comprising an amino acid sequence selected from:

[0267] Embodiment 19. The fusion polypeptide of any one of embodiments 1 to 18, wherein all of the amino acids in the fusion polypeptide are linked by amide bonds.

[0268] Embodiment 20. The fusion polypeptide of any one of embodiments 1 to 18, wherein at least two adjacent amino acids are linked by a non-amide bond.

[0269] Embodiment 21 The fusion polypeptide of any one of embodiments 1 to 20, wherein one or more of the amino acids in said polypeptide comprises a modification.

[0270] Embodiment 22. The fusion polypeptide of embodiment 21, wherein the modification comprises a maleimide group, a methyltetrazine group, a 3-nitro-pyridine-2-carboxylic acid group, a 1,4-bis(bromomethyl)-benzene group, a poly(ethylene glycol) group, a 5-carboxyfluorescein group, a nitropyridine group, a pyridyl disulfide, and a pyridine.

[0271] Embodiment 23. The fusion polypeptide of embodiment 21 or 22, comprising an amino acid sequence of formula IX: KLFEAIEGFIENGWEGMIDLWNX1X2YGRKKRRQRR (SEQ ID NO: 173), wherein X1, if present, is Gly and X2 is Cys(methyltetrazine) or Cys(3-nitro-2-pyridinesulfenyl).

[0272] Aspect 24. KLFEAIEGFIENGWEGMIDLWNC*YGRKKRRQRR (Peptide 87, SEQ ID NO:87) (where "C*" is Cys (methyltetrazine)); KLFEAIEGFIENGWEGMIDLWNGC*YGRKKRRQRR (Peptide 88, SEQ ID NO:88) (where "C*" is Cys (methyltetrazine)); KLFEAIEGFIENGWEGMIDLWNC*YGRKKRRQRR (Peptide 89, SEQ ID NO:89) (where "C*" is Cys(3-nitro-2-pyridinesulfenyl)); and KLFEAIEGFIENGWEGMIDLWNGC*YGRKKRRQRR (Peptide 90, SEQ ID NO:90) (where "C*" is Cys(3-nitro-2-pyridinesulfenyl)) 24. The fusion polypeptide of embodiment 23, comprising an amino acid sequence selected from:

[0273] 25. A peptide comprising an amino acid sequence of formula X: KLFEAIEGFIENGWEGMIDLWNGX1YGRKKRRQRRX2 (SEQ ID NO: 174), wherein X1 is Cys(methyltetrazine-PEG4-maleimide), Cys(maleimide), Lys(PEG 23 )2, Lys(3-nitro-pyridine-2-carboxylic acid), Lys(PEG 23 )2, Lys(PEG 23 23. The fusion polypeptide of embodiment 21 or 22, wherein X2 is Cys(3-nitro-2-pyridine-2-carboxylic acid), or Cys(1,4-bis(bromomethyl)-benzene), and X2 is Cys(3-nitro-2-pyridine-sulfenyl) or Lys(methyltetrazine-PEG4).

[0274] Embodiment 26. The fusion polypeptide of embodiment 25, comprising an amino acid sequence selected from the amino acid sequences of peptide f1, peptide f2, peptide f3, peptide f4, peptide f4, peptide f6, peptide f7, peptide f11, peptide f13, and peptide f14 shown in FIG. 30.

[0275] Embodiment 27. The fusion polypeptide of any one of embodiments 1 to 26, wherein at least two adjacent amino acids are linked by a linker comprising one or more ethylene glycol monomers.

[0276] Embodiment 28. The fusion polypeptide of embodiment 27, wherein the linker is a polymer comprising 2, 4, 6, or 8 ethylene glycol monomers.

[0277] Embodiment 29. The fusion polypeptide of embodiment 1, comprising any one of the amino acid sequences of peptides 19 and 40 to 60 shown in FIG. 1.

[0278] Embodiment 30 The fusion polypeptide of embodiment 1, comprising the amino acid sequence of any one of the peptides depicted in FIG. 30.

[0279] Embodiment 31. The fusion polypeptide of embodiment 1, which does not contain the amino acid sequence of any of the peptides depicted in Figure 1 and designated 1-18 or 21-27.

[0280] Embodiment 32. A composition comprising the cargo delivery fusion polypeptide of any one of embodiments 1 to 31.

[0281] Embodiment 33 The composition of embodiment 32, further comprising a cargo, said cargo comprising one or more of a nucleic acid, a polypeptide, and a ribonucleoprotein complex.

[0282] Embodiment 34 The composition of embodiment 33, wherein said cargo comprises a targeting moiety.

[0283] Embodiment 35. The composition of embodiment 32, comprising a nucleic acid comprising a nucleotide sequence encoding a gene product of interest.

[0284] Embodiment 36 The composition of embodiment 35, wherein said gene product of interest is an antigen.

[0285] Embodiment 37 The composition of embodiment 35 or embodiment 36, wherein said nucleic acid is a recombinant expression vector.

[0286] Embodiment 38 The composition of embodiment 37, wherein said recombinant expression vector is a recombinant viral vector.

[0287] Aspect 39. a) a CRISPR-Cas effector polypeptide, or b) i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides; A fusion polypeptide comprising 33. The composition of embodiment 32, comprising:

[0288] Embodiment 40. The composition of embodiment 39, comprising a CRISPR-Cas guide nucleic acid.

[0289] Embodiment 41. The composition of embodiment 39 or embodiment 40, comprising a donor DNA template.

[0290] Embodiment 42 The composition of any one of embodiments 39 to 41, wherein said CRISPR-Cas effector polypeptide is a Type II CRISPR-Cas effector polypeptide, a Type V CRISPR-Cas effector polypeptide, or a Type VI CRISPR-Cas effector polypeptide.

[0291] Embodiment 43 The composition of embodiment 40, wherein said CRISPR-Cas guide nucleic acid is RNA.

[0292] Embodiment 44 The composition of embodiment 43, wherein said CRISPR-Cas guide nucleic acid is a single-molecule guide RNA or a bi-molecule guide RNA.

[0293] Embodiment 45. A CRISPR-Cas effector fusion polypeptide comprising i) a CRISPR-Cas effector fusion polypeptide and ii) one or more nuclear localization signals. The composition of any one of embodiments 39-44, comprising:

[0294] Embodiment 46. A CRISPR-Cas effector fusion polypeptide comprising i) a CRISPR-Cas effector polypeptide and ii) one or more heterologous effector polypeptides. The composition of any one of embodiments 39-44, comprising:

[0295] Embodiment 47. The composition of embodiment 46, wherein at least one of said one or more heterologous effector polypeptides is a single-stranded nuclease, double-stranded nuclease, helicase, methylase, demethylase, acetylase, deacetylase, deaminase, integrase, recombinase, base editor, or prime editor.

[0296] Embodiment 48 The composition of any one of embodiments 39 to 47, wherein said CRISPR-Cas effector polypeptide or said CRISPR-Cas effector fusion polypeptide comprises a covalently linked targeting moiety.

[0297] Embodiment 49 The composition of embodiment 48, wherein said targeting moiety is Protein A, Protein G, an aptamer, a DARPin, or an antibody.

[0298] Embodiment 50. The composition of embodiment 49, comprising an antibody non-covalently bound to an affinity moiety.

[0299] Embodiment 51. The composition of embodiment 49 or embodiment 50, wherein said antibody specifically binds to an epitope on the surface of a eukaryotic cell, thereby targeting said composition to said cell.

[0300] Embodiment 52 The composition of any one of embodiments 39 to 51, wherein the CRISPR-Cas effector polypeptide or the CRISPR-Cas effector fusion polypeptide comprises a non-polypeptide polymer.

[0301] Embodiment 53 The composition of embodiment 52, wherein the non-polypeptide polymer is poly(ethylene glycol).

[0302] Embodiment 54. The composition of any one of embodiments 40 to 53, wherein said CRISPR-Cas effector polypeptide and said guide nucleic acid are present in a ribonucleoprotein (RNP) complex.

[0303] Embodiment 55. The composition of embodiment 54, wherein the molar ratio of cargo delivery fusion polypeptide to RNP is at least 3:1.

[0304] Embodiment 56. The composition of embodiment 55, wherein the molar ratio of cargo delivery fusion polypeptide to RNP is between 10:1 and 50:1.

[0305] Embodiment 57. The composition of any one of embodiments 32 to 56, wherein said cargo delivery fusion polypeptide is present in the composition at a concentration of from about 2 μM to about 50 μM.

[0306] Embodiment 58. The composition of any one of embodiments 32 to 57, further comprising one or more of a solubilizing agent, a surfactant, a buffer, a salt, and a protease inhibitor.

[0307] Embodiment 59. The composition of any one of embodiments 32 to 58, comprising poly(ethylene glycol), a non-ionic surfactant, or both.

[0308] Embodiment 60. A method for delivering a cargo to a target population of eukaryotic cells, comprising contacting said cells with a composition of any one of embodiments 32 to 59, thereby generating a modified target population of eukaryotic cells comprising said cargo.

[0309] Embodiment 61 The method of embodiment 60, wherein said target population of eukaryotic cells comprises T cells, stem cells, natural killer cells, renal cells, or neuronal cells.

[0310] Embodiment 62 The method of embodiment 60, wherein said target eukaryotic cell is a hematopoietic stem cell or hematopoietic progenitor cell.

[0311] Embodiment 63 The method of any one of embodiments 60 to 4362, wherein the cell is in vitro.

[0312] Embodiment 64 The method of embodiment 63, wherein after said contacting step, at least 50% of said target population of eukaryotic cells retain viability.

[0313] Embodiment 65 The method of embodiment 63 or embodiment 64, comprising a step of introducing a second composition comprising a second cargo into said modified target population of eukaryotic cells.

[0314] Embodiment 66 The method of embodiment 65, wherein said introducing step is by electroporation or transfection.

[0315] Embodiment 67 The method of embodiment 66, wherein said transfection comprises contacting said modified target population of eukaryotic cells with a recombinant viral vector.

[0316] Embodiment 68 The method of any one of embodiments 60 to 62, wherein the cell is in vivo.

[0317] 69. A method for delivering a DNA molecule to a eukaryotic cell, comprising: a) an amphipathic cargo-delivery fusion polypeptide, i) an endosomolytic polypeptide; and ii) a cell-penetrating polypeptide; and b) a DNA molecule comprising a nucleotide sequence encoding an immunogenic polypeptide. A composition comprising The method comprises the step of contacting the

[0318] Embodiment 70 The method of embodiment 69, wherein said immunogenic polypeptide is a viral polypeptide.

[0319] Embodiment 71. The endosomolytic polypeptide comprises: a) contains the amino acid sequence GLFEAIAEFIENGWEGLIEGWYG (SEQ ID NO: 163), b) containing 1 to 5 amino acid substitutions relative to GLFEAIAEFIENGWEGLIEGWYG (SEQ ID NO: 163), or c) comprises the amino acid sequence GLFEAIEGFIENGWEGMIDGWYG (SEQ ID NO: 164); or d) GLFEAIEGFIENGWEGMIDGWYG (SEQ ID NO: 164) containing 1 to 5 amino acid substitutions; The method of embodiment 69 or embodiment 70, wherein said endosomolytic polypeptide has a length of about 20 amino acids to about 30 amino acids.

[0320] Embodiment 72. The method of any one of embodiments 69 to 71, wherein the cell-penetrating polypeptide comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 207), YGRKKRRQRR (SEQ ID NO: 160), or GRKKRRQRRR (SEQ ID NO: 161), and has a length of 10 amino acids to 15 amino acids.

[0321] 73. A method for delivering a ribonucleoprotein (RNP) to a eukaryotic cell, comprising: a) an amphipathic cargo-delivery fusion polypeptide, i) an endosomolytic polypeptide; and ii) a cell-penetrating polypeptide; and b) an RNP, i) a CRISPR-Cas effector polypeptide; and ii) a guide nucleic acid; The RNP A composition comprising The method comprises the step of contacting the

[0322] 74. The endosomolytic polypeptide comprises: a) contains the amino acid sequence GLFEAIAEFIENGWEGLIEGWYG (SEQ ID NO: 163), b) containing 1 to 5 amino acid substitutions relative to GLFEAIAEFIENGWEGLIEGWYG (SEQ ID NO: 163), or c) comprises the amino acid sequence GLFEAIEGFIENGWEGMIDGWYG (SEQ ID NO: 164); or d) GLFEAIEGFIENGWEGMIDGWYG (SEQ ID NO: 164) containing 1 to 5 amino acid substitutions; The method of embodiment 73, wherein the endosomolytic polypeptide has a length of about 20 amino acids to about 30 amino acids.

[0323] Embodiment 75. The method of embodiment 73 or embodiment 74, wherein the cell-penetrating polypeptide comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 207), YGRKKRRQRR (SEQ ID NO: 160), or GRKKRRQRRR (SEQ ID NO: 161), and has a length of 10 amino acids to 15 amino acids.

[0324] Embodiment 76 The method of any one of embodiments 73 to 75, comprising the step of introducing a DNA donor template into said cell.

[0325] Embodiment 77 The method of embodiment 76, wherein said donor template is present in a recombinant viral vector.

[0326] Embodiment 78 The method of embodiment 77, wherein said recombinant viral vector is a recombinant adeno-associated viral vector.

[0327] Embodiment 79 The method of any one of embodiments 73 to 78, wherein the donor template comprises a nucleotide sequence encoding a polypeptide.

[0328] Embodiment 80 The method of embodiment 79, wherein the polypeptide is a chimeric antigen receptor comprising a single chain Fv or a nanobody specific for a cancer-associated antigen.

[0329] Embodiment 81 The method of any one of embodiments 73 to 80, wherein the eukaryotic cell is an immune cell.

[0330] Embodiment 82 The method of embodiment 81, wherein the immune cell is a T cell, a B cell, or a NK cell.

[0331] Embodiment 83 The method of any one of embodiments 73 to 82, wherein the eukaryotic cell is in vivo.

[0332] Embodiment 84 The method of any one of embodiments 73 to 82, wherein the eukaryotic cell is in vitro.

[0333] Mode Set C The above aspects, including the embodiments of the subject matter of the present invention, may be useful alone or in combination with one or more other aspects or embodiments.Without limiting the above description, certain non-limiting aspects of the present disclosure are presented below.As will be clear to those skilled in the art upon reading this disclosure, each of the individually numbered aspects can be used or combined with any of the preceding or succeeding individually numbered aspects.This is intended to give support for all such combinations of each aspect, and is not limited to the combination of aspects explicitly shown below.

[0334] Aspect 1: a) an endosomolytic polypeptide; b) a cell-penetrating polypeptide; A cargo delivery fusion polypeptide comprising: the fusion polypeptide has a length of about 32 amino acids to about 35 amino acids, any two adjacent amino acids are independently linked by an amide bond or a non-amide bond; i) a positively charged amino acid at the N-terminus; ii) a positively charged amino acid within 5 amino acids from the N-terminus, and iii) a positively charged amino acid at position 22 one or more of Optionally, the cargo-delivery fusion polypeptide, wherein the positively charged amino acid is Lys, His, or Arg.

[0335] Embodiment 2. The fusion polypeptide of embodiment 1, comprising an amino acid sequence of any one of formulas I-VIII.

[0336] Embodiment 3. Formula I: KLFEX1IEGFIENGWEX2MIDX3WX4GX5GRKKRRQRR (SEQ ID NO: 165), comprising the amino acid sequence X1 is A, R, or K; X2 is A or G; X3 is L or G; X4 is N or Y; The fusion polypeptide of embodiment 1, wherein X5, if present, is Y.

[0337] Aspect 4. KLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 19, SEQ ID NO: 19), KLFEAIEGFIENGWEGMIDGWYG GRKKRRQRR (Peptide 40, SEQ ID NO: 40), KLFERIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 44, SEQ ID NO: 44), KLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 45, SEQ ID NO: 45), KLFEAIEGFIENGWEAMIDGWYGYGRKKRRQRR (Peptide 46, SEQ ID NO: 46), KLFEAIEGFIENGWEGMIDLWYGYGRKKRRQRR (Peptide 47, SEQ ID NO: 47), KLFEAIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 48, SEQ ID NO: 48), KLFEAIEGFIENGWEAMIDLWYGYGRKKRRQRR (Peptide 53, SEQ ID NO:53), KLFEAIEGFIENGWEAMIDGWNGYGRKKRRQRR (Peptide 54, SEQ ID NO:54), KLFEAIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 55, SEQ ID NO:55), KLFEAIEGFIENGWEAMIDLWNGYGRKKRRQRR (Peptide 56, SEQ ID NO:56), and KLFEKIEGFIENGWEAMIDLWNGYGRKKRRQRR (Peptide 57, SEQ ID NO:57) The fusion polypeptide of embodiment 3, comprising an amino acid sequence selected from the group consisting of:

[0338] Embodiment 5. The fusion polypeptide of embodiment 1, comprising an amino acid sequence of formula II: X1LFEX2IEGFIENGWEGMIDGWYGYGRKKRRQRR (SEQ ID NO: 166), wherein X1 is R or G and X2 is R or K.

[0339] Aspect 6. RLFERIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 42, SEQ ID NO: 42); RLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 43, SEQ ID NO: 43), and GLFERIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 41, SEQ ID NO: 41) The fusion polypeptide of embodiment 5, comprising an amino acid sequence selected from the group consisting of:

[0340] Embodiment 7. The fusion polypeptide of embodiment 1, comprising an amino acid sequence of formula III: GLFEAIEGFIENGWEX1MIDX2WNGYGRKKRRQRR (SEQ ID NO: 167), wherein X1 is A or G and X2 is G or L.

[0341] Aspect 8. GLFEAIEGFIENGWEAMIDGWNGYGRKKRRQRR (Peptide 50, SEQ ID NO:50), GLFEAIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 51, SEQ ID NO:51), and GLFEAIEGFIENGWEAMIDLWNGYGRKKRRQRR (Peptide 51, SEQ ID NO:51) 8. The fusion polypeptide of embodiment 7, comprising an amino acid sequence selected from the group consisting of:

[0342] Embodiment 9. Formula IV: GLFEAIEGFIENGWEX1X2IX3LWYGYGRKKRRQRR (SEQ ID NO: 168), comprising the amino acid sequence X1 is A or G; X2 is L or M; The fusion polypeptide of embodiment 1, wherein X3 is D or E.

[0343] 10. Amino acid sequence: GLFEAIEGFIENGWEAMIDLWYGYGRKKRRQRR (Peptide 49, SEQ ID NO: 49), or GLFEAIEGFIENGWEGLIELWYGYGRKKRRQRR (Peptide 58, SEQ ID NO:58) 10. The fusion polypeptide of embodiment 9, comprising:

[0344] Embodiment 11. The fusion polypeptide of embodiment 1, comprising an amino acid sequence of formula V: GLFX1AIAX2FIX3NGWX4GLIX5GWYGGRKKRRQRRR (SEQ ID NO: 208), wherein each of Xi, X2, X3, X4, and X5 is independently a non-coded amino acid.

[0345] 12. Amino acid sequence: GLFαAIAαFIαNGWαGLIαGWYGGRKKRRQRRR (peptide 59, SEQ ID NO:59), or GLFαAIAαFIENGWEGLIDGWYGGRKKRRQRRR (Peptide 60, SEQ ID NO: 60) 12. The fusion polypeptide of embodiment 11, comprising:

[0346] Embodiment 13 The fusion polypeptide of embodiment 11 or embodiment 12, wherein each of X1, X2, X3, X4, and X5 is α-aminoadipic acid.

[0347] Embodiment 14. The fusion polypeptide of embodiment 1, comprising an amino acid sequence of formula VI: KLFEX1IX2X3FIENGWEGMIX4X5WX6GYGRKKRRQRX7 (SEQ ID NO: 170), wherein Xi is A or H, X2 is E or A, X3 is G or E, X4 is D or E, X5 is G or L, X6 is E, H, K, R, or N, and X7, if present, is R.

[0348] Aspect 15. KLFEAIEGFIENGWEGMIDLWEGYGRKKRRQRR (Peptide 62, SEQ ID NO: 62), KLFEAIEGFIENGWEGMIDLWHGYGRKKRRQRR (Peptide 63, SEQ ID NO: 63), KLFEAIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 64, SEQ ID NO: 64), KLFEAIEGFIENGWEGMIDLWRGYGRKKRRQRR (Peptide 65, SEQ ID NO: 65), KLFEAIEGFIENGWEGMIDLWNGYGRKKRRQR (Peptide 69, SEQ ID NO: 69), KLFEAIEGFIENGWEGMIELWNGYGRKKRRQRR (Peptide 71, SEQ ID NO: 71), KLFEAIAEFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 72, SEQ ID NO: 72), KLFEHIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 105, SEQ ID NO: 105), KLFEHIEGFIENGWEGMIDLWYGYGRKKRRQRR (peptide 107, SEQ ID NO: 107), and KLFEHIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 109, SEQ ID NO: 109) 15. The fusion polypeptide of embodiment 14, comprising an amino acid sequence selected from:

[0349] Embodiment 16. The fusion polypeptide of embodiment 1, comprising an amino acid sequence of formula VII: GLFEX1IX2X3FIENGWEGMIDX4WX5GYGRKKRRQRR (SEQ ID NO: 171), wherein Xi is R, H, A, or K, X2 is E or A, X3 is G or E, X4 is L or G, and X5 is N, Y, K, or E.

[0350] Aspect 17. GLFERIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 66, SEQ ID NO: 66); GLFEHIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 68, SEQ ID NO: 68); GLFEAIAEFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 70, SEQ ID NO: 70), GLFEKIEGFIENGWEAMIDGWYGYGRKKRRQRR (Peptide 73, SEQ ID NO: 73), GLFEKIEGFIENGWEGMIDLWYGYGRKKRRQRR (Peptide 74, SEQ ID NO: 74); GLFEAIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 75, SEQ ID NO: 31), GLFEAIEEFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 76, SEQ ID NO: 24), GLFEKIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 98, SEQ ID NO: 98); GLFEKIEGFIENGWEGMIDGWKGYGRKKRRQRR (Peptide 99, SEQ ID NO: 99); GLFEKIEGFIENGWEGMIDGWEGYGRKKRRQRR (Peptide 100, SEQ ID NO: 100), GLFEKIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 101, SEQ ID NO: 101), GLFEKIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 102, SEQ ID NO: 102), and GLFEKIEGFIENGWEGMIDLWEGYGRKKRRQRR (Peptide 103, SEQ ID NO: 103) 17. The fusion polypeptide of embodiment 16, comprising an amino acid sequence selected from:

[0351] Embodiment 18. The fusion polypeptide of embodiment 1, comprising an amino acid sequence of formula VIII: HLFEX1IEGFIENGWEGMIDX2WX3GYGRKKRRQRR (SEQ ID NO: 172), wherein Xi is A or K, X2 is G or L, and X3 is N, K, E, or Y.

[0352] Aspect 19. HLFEAIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 92, SEQ ID NO: 92), HLFEAIEGFIENGWEGMIDGWKGYGRKKRRQRR (Peptide 93, SEQ ID NO: 93), HLFEAIEGFIENGWEGMIDGWEGYGRKKRRQRR (Peptide 94, SEQ ID NO: 94), HLFEAIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 95, SEQ ID NO: 67), HLFEAIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 96, SEQ ID NO: 96), HLFEAIEGFIENGWEGMIDLWEGYGRKKRRQRR (Peptide 97, SEQ ID NO: 97), HLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 104, SEQ ID NO: 104), HLFEKIEGFIENGWEGMIDLWYGYGRKKRRQRR (Peptide 106, SEQ ID NO: 106), and HLFEKIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 108, SEQ ID NO: 108) 20. The fusion polypeptide of embodiment 18, comprising an amino acid sequence selected from:

[0353] Embodiment 20. The fusion polypeptide of any one of embodiments 1 to 19, wherein all of the amino acids in the fusion polypeptide are linked by amide bonds.

[0354] Embodiment 21. The fusion polypeptide of any one of embodiments 1 to 19, wherein at least two adjacent amino acids are linked by a non-amide bond.

[0355] Embodiment 22 The fusion polypeptide of any one of embodiments 1 to 21, wherein one or more of the amino acids in said polypeptide comprises a modification.

[0356] Embodiment 23. The fusion polypeptide of embodiment 22, wherein the modification comprises a maleimide group, a methyltetrazine group, a 3-nitro-pyridine-2-carboxylic acid group, a 1,4-bis(bromomethyl)-benzene group, a poly(ethylene glycol) group, a 5-carboxyfluorescein group, a nitropyridine group, a pyridyl disulfide, and a pyridine.

[0357] Embodiment 24. The fusion polypeptide of embodiment 22 or 23, comprising an amino acid sequence of formula IX: KLFEAIEGFIENGWEGMIDLWNX1X2YGRKKRRQRR (SEQ ID NO: 173), wherein X1, if present, is Gly and X2 is Cys(methyltetrazine) or Cys(3-nitro-2-pyridinesulfenyl).

[0358] Aspect 25. KLFEAIEGFIENGWEGMIDLWNC*YGRKKRRQRR (Peptide 87, SEQ ID NO:87) (where "C*" is Cys (methyltetrazine)); KLFEAIEGFIENGWEGMIDLWNGC*YGRKKRRQRR (Peptide 88, SEQ ID NO:88) (where "C*" is Cys (methyltetrazine)); KLFEAIEGFIENGWEGMIDLWNC*YGRKKRRQRR (Peptide 89, SEQ ID NO:89) (where "C*" is Cys(3-nitro-2-pyridinesulfenyl)); and KLFEAIEGFIENGWEGMIDLWNGC*YGRKKRRQRR (Peptide 90, SEQ ID NO:90) (where "C*" is Cys(3-nitro-2-pyridinesulfenyl)) 25. The fusion polypeptide of embodiment 24, comprising an amino acid sequence selected from:

[0359] 26. A peptide comprising an amino acid sequence of the formula X: KLFEAIEGFIENGWEGMIDLWNGX1YGRKKRRQRRX2 (SEQ ID NO: 174), wherein X1 is Cys(methyltetrazine-PEG4-maleimide), Cys(maleimide), Lys(PEG 23 )2, Lys(3-nitro-pyridine-2-carboxylic acid), Lys(PEG 23 )2, Lys(PEG 23 24. The fusion polypeptide of embodiment 22 or 23, wherein X2 is Cys(3-nitro-2-pyridine-2-carboxylic acid), or Cys(1,4-bis(bromomethyl)-benzene), and X2 is Cys(3-nitro-2-pyridine-sulfenyl) or Lys(methyltetrazine-PEG4).

[0360] Embodiment 27. The fusion polypeptide of embodiment 26, comprising an amino acid sequence selected from the amino acid sequences of peptide f1, peptide f2, peptide f3, peptide f4, peptide f4, peptide f6, peptide f7, peptide f11, peptide f13, and peptide f14 shown in FIG. 30.

[0361] Embodiment 28. The fusion polypeptide of any one of embodiments 1 to 27, wherein at least two adjacent amino acids are linked by a linker comprising one or more ethylene glycol monomers.Embodiment 29. The fusion polypeptide of embodiment 28, wherein said linker is a polymer comprising 2, 4, 6, or 8 ethylene glycol monomers.

[0362] Embodiment 30. The fusion polypeptide of embodiment 1, comprising any one of the amino acid sequences of peptides 19 and 40 to 60 shown in FIG. 1.

[0363] Embodiment 31 The fusion polypeptide of embodiment 1, comprising the amino acid sequence of any one of the peptides depicted in FIG. 30.

[0364] Embodiment 32. The fusion polypeptide of embodiment 1, which does not contain the amino acid sequence of any of the peptides depicted in Figure 1 and designated 1-18 or 21-27.

[0365] Embodiment 33. A composition comprising the cargo delivery fusion polypeptide of any one of embodiments 1 to 32.

[0366] Embodiment 34. A method of delivering a cargo to a target population of eukaryotic cells, comprising contacting said cells with the composition of embodiment 33, thereby generating a modified target population of eukaryotic cells comprising said cargo. EXAMPLES

[0367] The following examples are put forward to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. For example, standard abbreviations such as bp: base pair(s), kb: kilobase(s), pl: picoliter(s), s or sec: second(s), min: minute(s), h or hr: hour(s), aa: amino acid(s), kb: kilobase(s), bp: base pair(s), nt: nucleotide(s), im: intramuscular, ip: intraperitoneal, sc: subcutaneous may be used.

[0368] Materials and Methods In the examples below, the following materials and methods were used:

[0369] T cells Primary human CD4+ T cells were isolated from Leukopack (purchased from Stemcell Tech or Allcells Inc) PBMCs using a negative isolation method (Stem Cell Technologies). Cells were used fresh or stored frozen at -80°C. Cells were cultured at 1.0 × 10 6 Cells were suspended in medium (Xvivo-15 + 5% FBS + 55 μM β-mercaptoethanol (βME) + 10 μM N-acetylcysteine) at 10 cells / mL. Cells were stimulated for 48 hours with CD3 / CD28 Dynabeads (Gibco Cat. No. 11131D) at 1 bead / cell ratio, 200 U / mL human IL-2 (Proleukin), 5 ng / mL IL-7 (Peptrotech Cat. No. 200-07), 5 ng / mL IL-15 (Peptrotech Cat. No. 200-15). On the day of application of the Cas9 delivery complex, cells were removed from the beads, washed with medium, and repopulated with 1 × 10 fresh growth medium supplemented with 300 U / mL IL-2. 6 Once the Cas9 RNP delivery complexes were prepared, the cells were centrifuged at 300 × g, resuspended in OptiMEM, and plated in 96-well round-bottom culture plates at 200 × 10 3 cells / well.

[0370] NK cells Primary human NK cells were isolated from leukopacks (Allcells) of healthy blood donors using an NK cell isolation kit (STEMCELL, according to the manufacturer's instructions). Freshly isolated NK cells were cultured in X-VIVO15 medium (Lonza) containing 5% fetal bovine serum, 50 μM 2-mercaptoethanol, 10 mM N-acetyl L-cysteine ​​with IL-2 (1,000 U / ml) and MACSiBead particles preloaded with anti-human CD335 (NKp46) and anti-human CD2 antibodies (Miltenyi Biotec). Cells were cultured for 5 days, beads were removed by magnetic separation, and then co-incubated with peptide-delivered RNP or electroporated with the same protocol as T cells (200k cells + RNP in P3 buffer, pulse code EH-115). After treatment, cells were rescued by adding regular medium containing 1000 U / mL IL2 as described above. The medium was changed every 3 days.

[0371] B cells Primary adult blood cells from anonymous healthy human donors were purchased as leukapheresis packs from StemCell Technologies, Inc. Primary human B cells were isolated by negative selection using the EasySep Magnetic B Cell Isolation Kit (StemCell, Cat. No. 17954) based on the manufacturer's guidelines. After isolation, B cells were activated and cultured in 10% fetal bovine serum, 50 μM 2-mercaptoethanol, 100 ng mL -1 MEGACD40L (Enzo), 200ng mL -1 Anti-human RP105 (Biolegend), 500 U mL -1 IL-2 (UCSF Pharmacy), 50 ng mL -1 IL-10 (ThermoFisher), and 10ng mL -1 1 × 10 in IMDM medium (ThermoFisher) containing IL-15 (R&D Systems). 6 cells mL -1B cells were then cultured at 4 °C for 2 days. Prior to editing on day 2, B cells were harvested and treated with either electroporation or peptide co-incubation as indicated. B cells were then rescued with pre-warmed growth medium and cultured at 0.5–1.0 × 10 6 cells mL -1 Fresh medium and B cell activation cocktail were added every 2–3 days.

[0372] HSPC G-CSF and Plerixafor-mobilized primary human CD34+ HSPCs (Stem Cell Technologies) were thawed and cultured for 48 h with StemSpan CC110 cytokine cocktail in Stem Span SFEM II medium (Stem Cell Technologies). Cell density was maintained at 200,000–500,000 cells per mL. Cells were cultured at 20 × 10 per well in 80 μL of SFEM II. 3 Cells were seeded in 96-well round-bottom plates with 1.25x CC110. RNPs were diluted to 20 μL in SFEM II and incubated for 10 min at room temperature. Peptides were mixed with the RNP-SFEM II mix and incubated for 10 min at room temperature. The peptide-RNP-SFEM II mix was added to 20,000 HSPCs in a final volume of 100 μL per well of a 96-well round-bottom plate. In some experiments, the cell density was also increased to 100,000 HSPCS per 100 μL. After 48 h, cytokines were replaced. 100 μL of StemSpan SFEM II supplemented with StemSpan CC110 cytokine cocktail (STEMCELL Technologies) was added to each well 2 days after co-incubation.

[0373] Cell viability assay Cell viability after treatment compared to untreated cells was measured with the CellTiter-Glo assay (Promega G7570) according to the manufacturer's instructions, and luminescence was measured in a Spark plate reader.

[0374] peptide Peptides were prepared by solid-phase synthesis (CPC Scientific Peptide Company) and either lyophilized or stored as 10 mM dimethyl sulfoxide (DMSO) stocks in a desiccator at −20° C. The sequences of each peptide are shown in FIG.

[0375] Cas9 fusion protein Cas9 and fusion proteins were expressed in E. coli and purified by nickel affinity, ion exchange, and size exclusion chromatography as previously described (Rouet et al. (2018) J. Am. Chem. Soc. 140:6596). Purified proteins were concentrated to approximately 50 μM in 20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 10% (v / v) glycerol and stored at -80 °C. "Cas9-1×NLS" contains a C-terminal SV40 nuclear localization signal (NLS), and the "Cas9-3×NLS" construct (also referred to as "triNLS" in some texts and figures) contains an N-terminal cMycNLS and C-terminal SV40 and NP sequences (Wu et al. (2019) Nature Med. 25:776.). "Cas9-6xNLS" (also referred to as 4+2xNLS in some text and / or figures) contains 4x N-terminal SV40 NLS and 2x C-terminal SV40 NLS sequences (Staahl et al. (2017) Nat. Biotechnol. 35:431) and was stored in 25 mM sodium phosphate pH 7.25, 300 mM NaCl, 200 mM trehalose. "prA-Cas9-3xNLS" contains an N-terminal protein A domain that allows binding to the Fc domain of IgG and the same NLS configuration as "Cas9-3xNLS". The sequences of each fusion protein are shown in Figures 29A-28E.

[0376] sgRNA Single molecule guide RNA (sgRNA) was purchased from Synthego or IDT, and each lyophilized stock was dissolved in water. Prior to use, sgRNA was suspended in 20 mM HEPES pH 7.5, 150 mM NaCl, heated at 95°C for 5 minutes, and gradually cooled to room temperature over 25 minutes to refold. Each spacer sequence is shown in Figure 26 and Figure 32.

[0377] Formation of RNPs Each Cas9 protein was diluted to 10 μM in "RNP buffer" (20 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 2 mM MgCl2). sgRNA was diluted to 15 μM in 20 mM HEPES pH 7.5, 150 mM NaCl. Cas9 protein was mixed with guide RNA in equal amounts to obtain 5 μM RNP complex with a Cas9:guide RNA molar ratio of 1:1.5.

[0378] antibody Anti-CD3 antibody (OKT3, Invitrogen, 16-0037-85) was concentrated to 20 μM (Amicon Centrifugal Filter Units, UFC500396) and requantitated in Nanodrop. The control antibody ("IgG") was γ-globin from human blood (Sigma G4386) resuspended at 20 μM in phosphate buffered saline (PBS) pH 7.4. Anti-CD79b (CB3-1, BDBiosciences Cat. No. 555678), anti-CD22 (S-HCL-1, Biolegend Cat. No. 363502), anti-CD22 (HIB22 (Biolegend Cat. No. 302502), (anti-RP105MHR73-11, Biolegend Cat. No. 312913), and anti-CD71 (EPR4012, Abcam Cat. No. 108985) were concentrated to 6.7 μM (Amicon Centrifugal Filter Units, UFC500396) and requantified in Nanodrop.

[0379] Delivery complexes for co-incubation Cas9 RNP was mixed with antibody at a 1:1 molar ratio and allowed to bind for a minimum of 15 min at room temperature. Peptides in DMSO were diluted to 1 mM in HO and added to the RNP:Ab mix. Each complex was added to a 96-well round-bottom plate and cells in OptiMEM were added directly on top of the RNP / Ab / peptide complex (200 × 10 in 100 μL OptiMEM). 3 Cells were incubated for 1 h at 4°C for 1 h (cells / well). Final concentrations of RNP:Ab complex on cells were 500 nM and peptide was 10 μM unless otherwise stated. After 1 h of treatment, cells were split into two plates with each treatment, 50 μL per plate, and treatments were diluted by adding but not removing 150 μL of recovery medium (Xvivo15 + 7.5% fetal bovine serum (FBS) + 450 U / mL IL2) per well.

[0380] Cas9 RNP electroporation Cas9 RNP was electroporated (nucleofected) into T cells using a P3 Primary Cell 4D nucleofector (Lonza catalog no. V4XP-3032) with P3 buffer + supplements, using an EH-115 pulse code.

[0381] Continuous Editing At each successive editing treatment, stimulated CD4 + T cells were treated with 50 pmol of prA-Cas9-3×NLS complexed to the OKT3 antibody and combined with 10 μM A5K (peptide no. 22). The first treatment targeted β2M, and after 2 days the edited cells were pooled, mixed, and split into two groups, one group additionally underwent editing at the CD4 locus, while the other group remained as a single edited control group. After 2 days, the β2M and CD4 edited cell group was split into two groups, one group was edited for CD5, and the second group remained as a double (β2M and CD4) edited control set. After 2 days the cells were analyzed by flow cytometry to obtain KO levels of β2M, CD4, and CD5 in all treatment groups.

[0382] Editing neural precursor cells Neural progenitor cells (NPCs) were derived from Ai9 mice (Jackson Laboratories, stock number: 007909) that contain a loxP-flanked STOP cassette that inhibits transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato). SpCas9 guide RNAs as previously described were applied to cleave the STOP cassette and express TdTomato. Guides "A" and "B" were applied as paired guides to excise the cassette (Tabebordbar et al. (2016) Science 351:407) or guide "C" (also known as sg298 (Staahl et al. (2017) supra)) was applied as a sole guide. Spacer sequences are shown in Figure 26.

[0383] NPCs were cultured in DMEM / F12 with Glutamax supplemented with 10 mM HEPES pH 7.5, 1x NEAA (Fisher Sci 11-140-050), Pen-Strep (100U / mL) (Fisher Sci 15140-122), 1x B27 (Fisher Scientific 17504-044), and 1x N2 supplement (Thermo 17502048). Cells were seeded at a density of 15000 or 50000 cells per well in 96-well plates. Delivery complexes were applied to cells and incubated for 5 days before analysis for TdTomato signal by flow cytometry.

[0384] Non-viral knock-in (KI) DNA Oligonucleotide ("Oligo") Design and HDR Strategy: The HDR template for creating an N-terminal fusion of the extracellular portion of the CD5 molecule with FLAG was synthesized as a single-stranded 160-mer oligonucleotide containing a 40-base left homology arm and a 40-base right homology arm, and a truncated Cas9 binding site (Nguyen et al. (2020) Nat. Biotechnol. 38:44; and Shy et al. (2021) BioRxiv doi: 10.1101 / 2021.09.02.458799.). Five days after knock-in, cells were stained for CD5 expression and FLAG tag expression and analyzed on an AttuneNXT flow cytometer.

[0385] Targeted knock-in using AAV vectors Knock-in was performed in T cells using recombinant AAV6 containing homology arms targeting the start of the first exon of the TRAC locus (Eyquem et al. (2017) Nature 543:113). The cargo was 1928zCAR flanked by P2A sequences; after knock-in, the CAR was transcribed co-cistronically with the mRNA encoding TCRα, but translated as a separate protein, and the TCR remained unexpressed on the surface. The CAR contained the CD8A signal sequence, SJ25C1scFv, CD28 hinge, transmembrane and costimulatory domains, and CD3ζ signaling domain.

[0386] Knock-in by electroporation of RNP to knock out TCRα After 2 days of activation, T cells were detached from the Dynabeads, pelleted by centrifugation, and resuspended in LonzaP3 primary cell solution. TRACsgRNA (Synthego) and Cas9 protein (QB3 MacroLab, "6xNLS") were incubated at a 2:1 ratio (37°C, 15 min) and 60 pmol of RNP was added to 2 x 10 cells per well of an Amaxa nucleofector plate. 6The cells were electroporated using the Lonza nucleofection device and EH115 cell type protocol, and cells were incubated in serum-free medium at 2 × 10 6 The cells were then allowed to recover to 0.2 × 10 cells / ml and placed back in the tissue culture incubator for 1 hour. 6 cells (or 4 x 10 of the same concentration for multiplex editing experiments). 6 cells) at a multiplicity of infection of 5 × 10 4 After overnight incubation, the cells were split into two wells, pelleted by centrifugation, and the AAV-containing medium was replaced with fresh serum-containing medium.

[0387] Knock-in by co-incubation of RNP and A5K to knock out TCRα After 2 days of activation, T cells were detached from the Dynabeads, pelleted by centrifugation, and plated at 0.2 × 10 cells per well. 6 The cells were resuspended in Opti-MEM as 1x10 cells. 50 pmol of TRAC targeting RNP (sgRNA to Cas9 ratio = 1.2:1) and 1000 pmol of A5K (or 1500 pmol for sequential editing experiments) were added together and the cells were incubated with this mix to a volume of 100 μl in the well (tissue culture incubator, 1 h). The cells were treated with AAV at different time points -1 h, -30 min, +1 min, +30 min, and +1 h with respect to treatment with the RNP-peptide mixture, otherwise kept in the tissue culture incubator. The cells were then split into two wells and 50 μl or 150 μl of serum-free medium was added depending on the condition to obtain a volume of 1x10 cells, respectively. 6 cells / ml or 0.5 x 10 6 In the "wash" condition, cells were pelleted by centrifugation without splitting, and the supernatant was replaced with 100 μl of serum-free medium to give 2 × 10 6 The cells were treated with AAV and passaged in the same manner as in the electroporation conditions.

[0388] Flow cytometry Flow cytometry was performed on an Attune NxT flow cytometer equipped with a 96-well autosampler (ThermoFisher Scientific). Cells were resuspended in FACS buffer and stained with surface marker targeting antibodies and LIVE / DEAD stains according to the manufacturer's instructions. Analysis was performed using FlowJo.

[0389] Amplicon sequencing Genome editing was quantified by next-generation sequencing (NGS) of amplicons around the Cas9 target site. Cells were pelleted by centrifugation at 500 × g for 5 min and washed twice with PBS before being resuspended in 50 μL of Quick Extract (Lucigen) per well and incubated at room temperature for 20 min. Samples were then heated at 65 °C for 10 min and then at 95 °C for 5 min. Genomic DNA was stored at -20 °C. Polymerase chain reaction (PCR) amplification was performed using GXL polymerase according to the manufacturer's instructions. Amplicons were cleaned up by SPRI beads (UC Berkeley Sequencing Core) and quantified by Nanodrop.

[0390] Samples were deep sequenced on an Illumina MiSeq with 300bp paired-end reads to a depth of at least 10,000 reads per sample. Editing outcomes were analyzed using Cortado (https:( / / )github.com / staciawyam / cortado). Briefly, reads were adapter trimmed and then fused into a single read. These concatenated reads were then aligned to the target reference sequence to identify editing events at the cut site. NHEJ rates were calculated by counting reads with insertions or deletions overlapping the cut site or occurring within a 3-base pair window on either side of the cut site. SNPs occurring within the window around the cut site were not counted for NHEJ. The sum of NHEJ reads was then divided by the total number of aligned reads to obtain the NHEJ rate (%). For base editing experiments, concatenated reads were aligned to the target reference sequence to identify base editing events at the target locus. Base editing rates were calculated as the number of reads with edits relative to the total number of aligned reads, calculated for each site separately.

[0391] DNA Vaccines Plasmids For the cell reporter assay, we used plasmid DNA encoding the nanoluciferase gene on a CMV promoter, and for the in vivo mouse experiments, we used plasmid DNA encoding the RBD subunit of the SARS-CoV-2 spike protein as the vaccine antigen, also on a cytomegalovirus (CMV) promoter.

[0392] Cas9 and guide RNA The crRNAs (IDT) were designed to target several sites throughout the plasmid. RNPs were prepared using each crRNA and tracrRNA (IDT) with inactive Cas9 (Alt-R® Sp dCas9 Protein V3, IDT) such that the RNPs bind (but do not cleave) the plasmid DNA. The crRNAs and tracrRNAs were mixed in equimolar amounts, diluted to 12 μM in 20 mM HEPES pH 7.5, 150 mM NaCl, and heated at 37° C. for 30 minutes to form dual guide RNAs. The dCas9 was diluted to 10 μM in 20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 10% (v / v) glycerol, mixed in equal amounts with the dgRNA, and incubated at 37° C. for 15 minutes to give a final RNP of 5 μM and a Cas9 to dgRNA ratio of 1:1.2.

[0393] Formation of the delivery complex Plasmid DNA was mixed with Cas9 RNPs (molar ratio of each RNP = 1:1, 0–8 RNP binding sites per plasmid) to allow binding, then mixed with the indicated concentrations of peptide and applied to cells.

[0394] DC2.4 cell luciferase assay DC2.4 cells were cultured in DMEM containing Glutamax, 10% FBS, 1% NEAA, and 1% sodium pyruvate. Delivery complexes were applied to DC2.4 cells. After 24 or 48 hours, cell lysates were harvested using M-Per lysis reagent (ThermoFisher 78501) and luciferase expression was quantified using the Nano-Glo Luciferase Assay Kit (Promega, N1120) and measured on a Spark plate reader. Protein content of each sample was measured by Bradford protein assay with bovine serum albumin (BSA) standard as the reference. Luciferase expression was normalized to protein levels in each sample.

[0395] Mouse studies The delivery complex was injected intramuscularly into the hind leg of Balb / c mice on day 0 and again 2-3 weeks later. Further purified recombinant Spike protein antigen was administered intramuscularly (IM) 3-5 weeks after vaccination. Blood was collected at the indicated time points to assess serum levels of anti-Spike RBD IgG antibodies by ELISA, which have been shown to correlate highly with clinical protection against disease in clinical trials.

[0396] Cas12a protein Cas12a protein was purchased from IDT. Alt-RA.s.Cas12a Ultra (10001273) (modified Acidaminococcus species construct) (Zhang et al. (2021) Nat. Comm. volume 12, Article number: 3908).

[0397] Adenine Base Editing Protein The adenine base editor (ABE) protein contains a "nickase" version of CRISPR Cas9 from Streptococcus pyogenes (a D10A mutation that disables one catalytic center) fused to its N-terminus with a pair of TadA deaminase domains (whose N-terminal majority is catalytically disabled) from the fused evolved form "8e" (Richter et al. Nat. Biotechnol. (2020) 38:7), and NLS sequences added to the N- and C-termini of the ABE fusion protein. Three versions of nickase Cas9 are used, each with different protospacer adjacent motif (PAM) recognition properties: a wild-type NGG version, a modified NG version (Nishimasu et al. (2018) Science 361:6408), or a modified NRCH version (Miller et al. 2020 Nat Biotechnol. (2020) 38:4). The ABE protein was expressed in E. coli and purified by nickel affinity chromatography, ion exchange chromatography, and size exclusion chromatography as previously described (Rouet et al. (2018) J. Am. Chem. Soc. 140:6596.). The amino acid sequence is shown in Figure 27 and Figure 31.

[0398] Targeted knock-in using AAV vectors In primary human B cells and NK cells, knock-in was performed using a recombinant AAV6 containing homology arms targeting the start of the first exon of the CLTA locus, generating an N-terminal fusion of sfGFP to clathrin (Source: Roth et al. 2018 Nature 549:405).

[0399] Knock-in by co-incubation of RNP with A5K to knock-in sfGFP-clathrin fusions For sfGFP-CLTA knock-in in B cells, B cells were cultured as above for 2 days, pelleted by centrifugation, and cultured in Opti-MEM at 0.2 × 10 6For sfGFP-CLTA knock-in in NK cells, NK cells were cultured as above for 5 days, removed from the beads by magnetic separation, pelleted by centrifugation, and resuspended in Opti-MEM at 0.2 × 10 6 Cells were resuspended in 1000 μl / well. For both B and NK editing, 50 pmol of CLTA targeting RNP (sgRNA:Cas9 ratio = 1.2:1) was combined with 1000 pmol of A5K and cells were incubated with this mix to a volume of 100 μl in the well (tissue culture incubator, 1 h). Cells were then treated with AAV at a multiplicity of infection of 5 x 10^4. After overnight culture, cells were split into two wells, pelleted by centrifugation, and the AAV-containing medium was replaced with fresh serum-containing medium.

[0400] In vivo editing of human T cells in humanized (PBMC NSG) mice NOD SCID gamma (NSG) mice (Shultz et al. (2007) Nat. Rev. Immunol. 7:118) injected with peripheral blood mononuclear cells (PBMCs) from a single human donor were administered the prepared formulations by tail vein injection at 6–8 weeks of age. CRISPR-Cas9 RNP was prepared to contain an sgRNA targeting the β2M locus paired with prA-Cas9-triNLS. 500 pmol of RNP was mixed with either 100 pmol of Fc-silenced CD3 (OKT3) antibody or no antibody, and 5 nmol of either peptide 1 (E5-TAT) or peptide number 22 (A5K) in all formulations with 0.05% Tween-80. This resulted in four samples containing RNP: RNP and peptide 1 ± OKT3, and RNP and peptide 22 ± OKT3. Each RNP preparation was filtered using a 0.22um sterile filter prior to administration. Animals were sacrificed 7 days after injection, splenocytes were isolated, and editing efficiency was assessed by amplicon-based deep sequencing. PCR primers used for amplicon generation were specific for the human genome; no amplicons were generated when mouse genomic DNA was included as template. Some splenocyte samples were sorted by fluorescence-activated cell sorting (FACS) to identify CD5 + T cells and CD5 - Non-T cells (mainly B cells) were isolated and indels were detected by amplicon-based deep sequencing.

[0401] In vivo editing of mouse brains 7 μL of RNP formulations were stereotaxically injected into the striatum of Ai9 mouse brains (coordinates: AP: +0.5 mm relative to bregma, MR and ML relative to midline: 2.0 mm, DV: 2.5 mm) at an injection rate of 1 μL / min using convection-enhanced delivery (CED). The left striatum was injected with the RNP-tween formulation, and the right striatum was injected with the RNP-peptide-tween formulation (peptide number 55). Each RNP sample contained triNLS SpCas9 protein, underlying the reported RNP concentrations, complexed with 0.75-fold molar equivalents of Ai9 "A" gRNA and Ai9 "B" gRNA (cumulatively resulting in a 1.5-fold molar excess of gRNA over Cas9 protein). These two gRNA sequences can produce two coordinated DNA cleavages to excise a repressor cassette that activates downstream tdTomato expression. In the 7 μL formulation injected, the RNP concentration was 54.4 μM in samples with peptide 55 and 52.6 μM in samples without peptide 55, the peptide 55 concentration was 2 mM, and the Tween-80 concentration was 0.05%. Mice were 10 weeks old at the time of delivery, and three male and two female mice were injected. Brains were harvested 21 days after formulation administration. Tissue sections were stained for NeuN (neurons, chicken anti-NeuN from GeneTex), DAPI (nuclei), and tdTomato (polyclonal rabbit anti-RFP from Invitrogen). For highly edited regions, at least three 20x confocal regions of interest per animal were imaged for quantification using a Dmi8 fluorescent microscope. Merged 20x confocal images were used to image tdTomato using QuPath software. + The percentage of neurons was quantified.

[0402] Transcriptome analysis CD4 +T cells were edited with Cas9-RNP targeting AAVS1 as described above. RNA was extracted from treated cells (nt, DMSO, A5K, electroporation) at three time points (6 h, 1 day, 7 days after editing) from four donors using the RNeasyMicro kit (Qiagen, Cat. No. 74004), quantified using NanoDrop, and stored at -80°C. RNA samples were submitted to the UCSF-HDFCCC Laboratory for Cell Analysis Shared Resource Facility for nCounter analysis using the NanoString CAR-T Characterization Panel. Volcano plot gene expression fold changes and Benjamini-Yekutieli adjusted p-values ​​were obtained using nSolver software. For each treatment comparison of interest, nSolver analysis was performed for each time point separately and for all time points incorporated. The latter approach used more data as input, was more sensitive in identifying significantly affected genes, and was less biased towards time point specific results.

[0403] Translocation analysis Cells were pelleted by centrifugation at 300×g for 5 min, resuspended in 40 μL of QuickExtract solution (Lucigen) per well and incubated at 65°C for 10 min, followed by 95°C for 5 min. Genomic DNA was stored at -20°C. Primers and 5'6-FAM labeled probes were synthesized by IDT (ZEN-3'Iowa Black FQ quencher). Droplet digital PCR reactions were prepared using ddPCR Supermix for Probes (no dUTP) (Bio-Rad, Cat. No. 1863023) and droplets were generated and read using a QX200 droplet generator and reader. Data were analyzed using Bio-Rad QuantaSoft Analysis Pro software.

[0404] Example 1 Peptide Screening Peptides 1–60 (Figure 1) were immunoreacted with human primary CD4 +We screened for Cas9-mediated KO at the β2M locus in T cells. Peptides 1-58 (Figure 1) were + We screened for Cas9-mediated NHEJ at the erythroid-specific BCL11a enhancer locus in HSPCs. We evaluated the effects of non-ionic detergents on peptide-mediated genome editing. The results are shown in Figures 3-8.

[0405] Figure 3. Screening of peptides 1-37 for Cas9-mediated KO at the β2M locus in human primary CD4+ T cells as measured by flow cytometry. prA-Cas9-1×NLS with OKT3 was combined with each peptide at a concentration of 5 μM or 10 μM. DMSO control is RNP (but without peptide) mixed with DMSO at the concentration introduced by the peptide stock solution. This screen demonstrates that specific single amino acid changes from the original E5-TAT (peptide no. 1) or INF7-TAT (peptide no. 11) can result in significantly higher delivery efficiency, as determined by the resulting editing rate.

[0406] As shown in Figure 3, different peptides can edit primary human CD4+ T cells to various degrees when co-incubated with Streptococcus pyogenes Cas9 ribonucleoprotein (RNP) complexes targeting the β2M locus. Peptide-mediated delivery of Cas9 RNP cargo was effective at final peptide concentrations of either 5 μM or 10 μM. In detail, peptide 19 (G1K variant of INF7-TAT) edits T cells much more efficiently than INF7-TAT. This experiment was performed in the absence of targeting antibodies and used the "1NLS" Cas9 construct, which is less effective than Cas9 constructs with more fused NLS sequences.

[0407] Figure 4A and Figure 4B. Screening of peptides for Cas9-mediated KO at the CD4 locus in human primary CD4+ T cells assessed by flow cytometry (panel A) or deep sequencing (panel B). prA-Cas9-3xNLS was combined with no IgG or an equal amount of a control non-targeting heterologous IgG mixture (ctrl IgG) or an anti-CD3 targeting antibody (OKT3) and applied to cells with peptides at a final concentration of 10 μM. Condition "19&30" is an equimolar mixture of peptides 19 and 30. Enhancement of OKT3-mediated editing varies depending on the peptide used. Replicate experiments represent the results of two different human cell donors.

[0408] As shown in Figures 4A and 4B, different peptides can edit primary human CD4+ T cells to different degrees when co-incubated with S. pyogenes Cas9 ribonucleoprotein (RNP) complexes targeting the CD4 locus. Peptide-mediated delivery of Cas9 RNP cargo (sometimes used in combination with antibodies) is effective and can be enhanced by the inclusion of cell-targeting antibodies. In this example, the Cas9 construct features a fusion with an IgG-binding protein A domain and three NLS sequences. This Cas9 construct can be bound to the anti-CD3 mAb OKT3, which is known to induce endocytosis into T cells via interaction with the T cell receptor. The presence of OKT3 can enhance peptide-mediated editing efficiency, as shown by the bars in grey, which often show significantly higher editing rates than the bars without IgG (black) or with ctrl IgG (white).

[0409] Figure 5A and Figure 5B. (A) Screening of peptides no. 40-60 (and a selection of comparison peptides from the screening of "parent peptides" no. 1-37) for Cas9-mediated KO at the β2M locus in human primary CD4+ T cells, measured by flow cytometry. prA-Cas9-1xNLS was combined with anti-CD3 antibody OKT3 and applied to cells together with peptides at a concentration of 10 μM. (B) Live cell counts from each treatment condition measured by live / dead fixed violet staining (Thermofisher) and flow cytometry. Replicates are from two different human cell donors. In both (A) and (B), "No pep" = Cas9 without peptide, "NT" = cells not treated with Cas9 or peptide.

[0410] As shown in Figures 5A and 5B, further peptide screening showed that some (but not all) of the peptide sequences with sequence elements derived from "parent peptide" numbers 1-37 promoted genome editing in T cells, and the editing was more efficient than that mediated by either parent peptide and more efficient than that mediated by electroporation. Importantly, after treatment with Cas9 and peptide, the viability of T cells was close to that of "no peptide" and "untreated" control conditions, and the viability was superior compared to electroporated cells (peptide number 31 was the exception).

[0411] Figure 6. Screening of peptides no. 1-37 for enabling Cas9-mediated NHEJ at the erythroid-specific BCL11a enhancer locus in CD34+ HSPCs as measured by deep sequencing at concentrations of 5 μM or 10 μM. The results of this initial screen indicate that specific amino acid changes from the original E5-TAT (peptide no. 1) or INF7-TAT (peptide no. 11) can achieve highly efficient delivery as measured at the editing level.

[0412] As shown in Figure 6, primary human CD34+ HSPCs can be edited to various degrees when different peptides are co-incubated with the Cas9 ribonucleoprotein (RNP) complex of Streptococcus pyogenes targeting the erythroid-specific Bcl11a enhancer locus. Peptide-mediated delivery of the Cas9 RNP cargo was effective at final peptide concentrations of either 5 μM or 10 μM. In detail, peptide 19 (G1K variant of INF7-TAT) edits CD34+ HSPCs much more efficiently than INF7-TAT. This experiment was performed in the absence of targeting antibodies and used a Cas9 construct fused with three NLS sequences.

[0413] Figure 7A-7C. (A) Screening of peptides no. 40-58 for promoting Cas9-3×NLS-mediated NHEJ in HSPCs (and comparison with peptides selected from the first round of screening) as measured by deep sequencing at a final peptide concentration of 5 μM or 10 μM. NHEJ at the erythroid-specific BCL11a enhancer locus in CD34+ HSPCs was detected by amplicon-based NGS. (B) HSPC viability under each condition as measured by CellTiter-Glo assay (Promega). (C) Composite score for each peptide calculated as [(viability (%) × NHEJ (%)) × 100]. Boxed = overall best peptide with a good balance between activity and toxicity. Note that E5-TAT is peptide no. 1 (Figure 1).

[0414] As shown in Figures 7A-7C, further peptide screening showed that some (but not all) peptides with sequence elements derived from "parent peptide" numbers 1-37 promoted genome editing in HSPCs more efficiently than the editing mediated by either parent peptide. Peptide number 55 is an example of a peptide that promoted efficient genome editing and maintained cell viability.

[0415] Figures 8A-8C. Effect of non-ionic surfactant additives on peptide-Cas9 formulations. (A) Peptide-mediated genome editing of human primary T cells in the absence or presence of 0.05% (w / v) Tween-80 (concentrations shown are the concentrations in the RNP formulation before application to cells and media). Cas9 RNPs targeted for CD4 knockout were precomplexed with either negative control, non-targeting IgG, or anti-CD3, which can promote endocytosis into T cells. Delivery (and genome editing) was enhanced by the inclusion of 5 μM (final concentration) of E5-TAT (peptide no. 1), G20L (peptide no. 30), or A5K (peptide no. 22). Knockout of CD4 was detected by flow cytometry. (B) Dynamic light scattering (DLS) was used to measure the average particle size of samples of the various formulations in panel (A). (C) Representative data from the OKT3+A5K, 0.05% Tween-80 sample shown in panels (A) and (B).

[0416] As shown in Figures 8A-8C, each peptide is able to mediate delivery of Cas9 RNP for efficient genome editing of human primary T cells, even in the presence of non-ionic detergents such as Tween-80™. This is important because Tween-80 can promote particle size reduction that may be important for tissue distribution in in vivo applications. A5K (Peptide No. 22) was able to retain full potency as part of a formulation containing 0.05% Tween-80, and such samples exhibited a predominant particle size of 10-20 nm, consistent with the 3D structure of the Cas9 RNP.

[0417] Example 2 Gene editing We tested the effects of different peptides (Figure 1) on gene editing in different cell types, and the results are shown in Figures 9-19.

[0418] Figure 9. Screening of Cas9-mediated genome editing in primary mouse neural progenitor cells (NPCs) from Ai9 mice, as measured by tdTomato signal detected by flow cytometry. Peptides E5-TAT (peptide 1) and A5K (peptide 22) at 10 μM ("high") or 5 μM ("low") were examined in combination with Cas9 3×NLS or Cas9(4+2×NLS) using either equal amounts of A / B guides targeting two loci, or C guides targeting one locus. In some conditions, Tween-80 was also added to the RNP and peptide formulations as an additive at a concentration of 0.05% (w / v). Cells were seeded at 50,000 cells per well. Flow cytometry was performed 5 days after cells were treated.

[0419] As shown in Figure 9, co-incubation of the peptides promoted genome editing in primary mouse neural progenitor cells. Peptide number 22 (A5K derivative of INF7-TAT) promoted editing more efficiently than peptide number 1 (E5-TAT). The cells were derived from Ai9 reporter mice, which express the fluorescent protein tdTomato when genome editing forms a small break that allows gene expression. Each peptide can mediate delivery of Cas9 RNP (and genome editing) even in the presence of the non-ionic detergent Tween-80 (also known as polysorbate 80), which can prevent peptide-mediated aggregation and promote the formation of a monodisperse solution of Cas9 RNP.

[0420] Figure 10. Screening of Cas9-mediated genome editing in primary mouse neural progenitor cells (NPCs) from Ai9 mice as measured by tdTomato signal by flow cytometry. Peptides A5K (peptide no. 22) and P55 (peptide no. 55) at a final concentration of 10 μM were used in combination with Cas9-(4+2) formed as RNP with A and B guides (equimolar mixture) to derepress the tdTomato locus and allow its expression when editing occurs. In some conditions, Tween-80 was also added to the RNP and peptide formulations as an additive at a concentration of 0.05% (w / v). Cells were seeded at 15,000 cells per well. Flow cytometry was performed 5 days after cells were treated.

[0421] As shown in Figure 10, co-incubation of the peptides promoted genome editing in primary mouse neural progenitor cells. Peptide number 55 (an INF7-TAT derivative with three amino acid substitutions (G1K, G20L, Y22N)) promoted editing more efficiently than peptide number 22 (an A5K derivative of INF7-TAT) when an equivalent amount of Cas9RNP was used. The cells were derived from Ai9 reporter mice, which express the fluorescent protein tdTomato when genome editing forms a small break that allows gene expression. Each peptide mediated delivery of Cas9 RNP (and genome editing) even in the presence of the non-ionic detergent Tween-80 (also known as polysorbate 80), which may prevent peptide-mediated aggregation and promote the formation of a monodisperse solution of Cas9 RNP. In this experiment, Tween-80 improved editing efficiency compared to the respective "no additive" conditions.

[0422] Figure 11. Editing of the CD45 locus in B, T and NK cells by peptide co-incubation or electroporation as measured by flow cytometry for CD45KO. Peptide-based delivery was performed using Cas9-6xNLS ("6xNLS") or Cas9-3xNLS ("triNLS") in combination with A5K peptide at a final concentration of 10 μM, or electroporation was performed according to the standard protocol. T and B cells were split every 2 days until CD45KO was read on an Attune NxT flow cytometer 6 days after delivery. NT-untreated. NTe-cells electroporated but without Cas9.

[0423] As shown in Figure 11, peptide-mediated delivery is effective in genome editing primary human B cells, T cells, and NK cells. Application of A5K peptide (10 μM final concentration) together with Cas9-6×NLS or Cas9-3×NLS resulted in highly efficient editing in primary human B cells (77% CD45 KO rate with Cas9-3×NLS). Slightly lower efficiency was observed in T cells (55% CD45 KO rate with Cas96×NLS). Under these conditions, NK cells were more resistant to peptide-based editing, with a 16% CD45 KO rate observed with Cas9-6×NLS.

[0424] Figures 12A and 12B. (A) Levels of B2M (β2M) KO in T and B cells cultured separately or as co-cultures of T and B cells, as measured by flow cytometry. Cells were treated with Cas9RNP and either A5K peptide (peptide #22) or E5-TAT peptide (peptide #1). For treatment with targeting antibodies, prA-Cas9 RNP was complexed with either non-targeting control IgG, OKT3, or anti-CD79b and mixed with E5-TAT peptide. Co-incubation conditions were compared to electroporated RNP ("E-KO") or cells electroporated without treatment as a negative control. (B) Editing ratios of T and B cells in several treatment conditions.

[0425] As shown in Figures 12A and 12B, peptides E5-TAT (Peptide No. 1) or A5K (Peptide No. 22) promoted Cas9-mediated genome editing of human primary T cells and human primary B cells when these two cell types were co-present in the same solution.

[0426] 13A-13C. (A) Percentage of primary human B cells exhibiting KO of β2M under different treatment conditions. prA-Cas9-3×NLS RNPs were complexed with antibodies (control non-targeting IgG, OKT3, anti-CD79b, anti-CD22-HCL, anti-CD22-HIB, RP1-5, anti-CD71) and mixed with E5TAT (peptide no. 1, dashed line) at a final concentration of 10 μM or A5K (peptide no. 22) at a final concentration of 10 μM without antibody ("none") and applied to cells. β2M levels were measured by flow cytometry 3 days after treatment. "KOe" refers to knockout mediated by Cas9 RNP delivered by electroporation. (B) Representative flow scatter plots showing the gating strategy for analyzing KO of β2M under the four conditions. (C) Viable cell counts of cells 3 days after treatment measured by flow cytometry. NT = non-treated cells. Repeat experiments are from two different human donors.

[0427] As shown in Figures 13A-13C, when each peptide was co-incubated with Cas9RNP to target and knock out the β2m gene, the peptides mediated efficient editing of human primary B cells. Peptide-mediated delivery of Cas9RNP resulted in editing rates comparable to those obtained with electroporation-mediated delivery of Cas9RNP, and the overall cell yield (number of edited cells obtained) was not significantly different between the two methods.

[0428] Figures 14A-14C. HDR knock-in of Flag tag into CD5 locus of primary human CD4+ T cells. Biological replicates from two human donors. "Tri-OKT3" = "prA-Cas9-3xNLS" with OKT3 antibody. "6xOKT3" = Cas9-6xNLS with OKT3 antibody. "Tri-IgG" = "prA-Cas9-3xNLS" with non-targeting control IgG. "6xIgG" = Cas9-6xNLS with non-targeting control IgG. "Tri-none-KO" = prA-Cas9-3xNLS without antibody and no DNA to be KI'd. "6xnone KO" = Cas9-6xNLS without antibody and no DNA to be KI'd. "NT" = untreated control cells. Figure 14A: Percentage of T cells positive for Flag tag under various conditions. Figure 14B: Representative flow plots from two conditions showing the gating strategy for quantifying KI of FlagTag in T cells. Figure 14C: Cell counts of CD5+Flag+ cells.

[0429] As shown in Figures 14A-14C, each peptide mediated delivery of the Cas9 RNP nuclease and the "donor" template DNA required to mediate knock-in editing in primary human T cells. The Cas9 RNP was delivered together with a template DNA molecule encoding a Flag tag. Using peptide-mediated delivery (without viral vector or electroporation), knock-in editing rates of approximately 5% were obtained.

[0430] Figures 15A-15C show knock-in of 1928z-CAR at the TCR locus in CD4+ T cells, where TRAC RNP was delivered by co-incubation with A5K peptide to KO TRAC, and the CAR locus was delivered by AAV6 at four different time points (30 min before, at the same time, 30 min after, or 2 h after RNP treatment). The co-incubation method was applied to the electroporation method ("KOe"), showing comparable CAR knock-in rates. Cells were analyzed for the presence of CAR by flow cytometry and plotted as (A) CAR+ cell counts, or (B) percentage of cells negative for TCR and positive for CAR, indicating KO, (C) representative scatter plots from flow cytometry. Data points represent biological replicates from two different human donors.

[0431] As shown in Figures 15A-15C, peptide-mediated delivery of Cas9 RNP can be used in combination with an AAV viral vector carrying a "donor" template DNA. In this case, the Cas9 RNP nuclease was targeted to the TRAC locus to form a double-stranded break. The AAV carried a template DNA sequence for a chimeric antigen receptor (CAR). When both were delivered to the same cell, it became possible to knock-in the CAR into the native TRAC locus, resulting in precisely engineered CAR-T cells with endogenous regulation of the CAR. When AAV was applied to cells simultaneously with the RNP peptide formulation, or 30 min before or after, CAR-T cells were efficiently generated (without the need for electroporation).

[0432] Figures 16A-16C. Sequential editing of CD4+ primary T cells at three genomic loci (TRAC, CD5, B2M) by co-incubation of Cas9 RNP (50 or 100 pmol, first number in ## / ## abbreviation) with 10 μM, 15 μM, or 20 μM A5K peptide (second number in ## / ## abbreviation). (A) Percentage of T cells edited to produce gene knockout (KO) at the TRAC locus, CD5 locus, or β2M locus. Two different electroporation conditions were used for comparison: Wilson:e, electroporation conditions close to the co-incubation conditions, and Marson:e, electroporation conditions fully optimized in the Marson lab. (B) Percentage of T cells edited to produce gene knockout (KO) at the TRAC locus, TRAC and CD5 loci, and TRAC, CD5, and β2M loci. (C) Flow cytometry scatter plots comparing triple-edited cells (top row) at the TRAC, CD5, and β2M loci with untreated cells.

[0433] As shown in Figures 16A-16C, sequential multiplex genome editing can be performed using peptide-mediated delivery of Cas9 RNP for genome editing in T cells. In this example, a formulation of Cas9 RNP and A5K peptide was applied to T cells every 2 days, with each Cas9 RNP targeting a different gene for KO (first TRAC, then CD5, and finally β2M). Since more cells survived each peptide-Cas9 treatment (compared to electroporation), a stable cell yield (including triple-edited cells) was obtained at the end of the experiment. In contrast, sequential electroporation yielded very few edited cells after two rounds of editing and almost none after three rounds of editing.

[0434] Figures 17A-17B. Peptide-mediated editing in T cells under various stimulation conditions. Peptide-mediated genome editing of primary human T cells previously treated with cytokines (but not activated) was assessed by flow cytometry for knockout levels of β2M. Three peptides were examined: E5-TAT, A5K, and peptide number 55, all at a final concentration of 10 μM in the medium. prA-Cas9-3×NLS RNP was complexed with either no antibody, control non-targeting IgG, anti-CD3 antibody, or anti-CD3 antibody and anti-CD28. Replicates are from two human donors. T cells were either kept in medium containing low concentrations of IL-2 (50 U / mL) for 48 hours until the delivery complex was added (FIG. 17A), or treated with a mixture of three cytokines at normal concentrations (IL-2 (200 U / mL), IL-7 (5 ng / mL), and IL-15 (5 ng / mL)) for 48 hours until the delivery complex was added (FIG. 17B).

[0435] We performed peptide-based delivery of prA-Cas9-3xNLS RNP into T cells that had not undergone bead-based activation. We tested the effects of two different low-level stimulation conditions, treating cells with low concentrations of IL-2 (Figure 17A) or a mixture of three cytokines (Figure 17B). As shown in Figures 17A-17B, editing of non-activated T cells was observed under all conditions, albeit at lower levels than with bead-based cell activation.

[0436] Figures 18A-18D. Knock-in of 1928z-CAR at the TCR locus in CD3+ bulk T cells followed by sequential KO. Cas9RNP targeting TRAC was delivered by electroporation or co-incubation with A5K peptide (peptide number 22) to KO TRAC, and 1 h later an AAV6 vector was applied to deliver the DNA HDR template encoding the CAR cassette. Two days later, a portion of the edited cells was edited to induce knockout of the β2M locus (CAR+B2M-), either by electroporation or co-culture. Two days later, another portion of the edited cells was edited to induce knockout of the CD5 locus (CAR+B2M-CD5-). The frequency of successfully edited cells (Figure 18A) and total cell number yield (Figure 18B) are reported as percentages for both electroporation and co-culture conditions. The percentage values ​​reported reflect cells with all edits made to a particular subset of cells. Four million cells were used for each experimental condition (electroporation or co-culture). (Figure 18C and Figure 18D) Sequential knockout at TRAC, β2M, and CD5 loci performed as in Figures 18A and 18B, but without the inclusion of AAV6 viral vector (i.e., no knock-in of CAR). As in Figures 18A and 18B, the frequency of successfully edited cells (Figure 18A) and total cell number yield (Figure 18B) are reported as percentages for both electroporation and co-culture conditions. Replicates are from three human donors.

[0437] As shown in Figures 18A-18D, sequential editing by peptide-mediated co-incubation of Cas9 RNP in primary human CD3+ T cells results in a similar percentage frequency of edited cells but a much higher yield (absolute number of edited cells) compared to sequential editing by electroporation.

[0438] Figures 19A-19D. Knock-in of 1928z-CAR at the TCR locus in CD3+ bulk T cells or cells treated for KO only (no KI with AAV) followed by sequential KO. Comparison of T cell phenotype between sequential editing by electroporation (Figure 19A and Figure 19C) and sequential editing by peptide co-incubation (Figure 19B and Figure 19D) in CD4+ or CD8+ T cells. All cell populations were assessed for CD62L and CD45RA phenotypes regardless of the edited phenotype.

[0439] As shown in Figures 19A-19D, sequential editing by electroporation reduces the percentage of naive T cells (CD62L+CD45RA+) present in both the CD4+ and CD8+ populations. Sequential editing by RNP / peptide co-incubation does not significantly affect the percentage of naive T cells (CD62L+CD45RA+) present in the CD4+ or CD8+ populations.

[0440] Example 3. Base editing Peptides were used to deliver RNP complexes containing Cas effector / base editor fusion proteins. The amino acid sequences of the Cas effector / base editor fusion proteins are shown in Figures 26A, 27A, 26C, and 26C. The results are shown in Figures 20, 21, and 22.

[0441] Figure 20. Adenine to guanine base editing at the CCR5 locus in primary human T cells delivered by peptide co-incubation with A5K peptide. Dose optimization was performed by applying 50, 100, or 200 pmol of SpCas9-NG or SpCas9-NRCH PAM variants to cells with 20 μM (final concentration) of A5K peptide. Cells were treated with a single dose or a second dose 2 days after the first dose. The addition of trehalose, a stabilizing additive, to the buffer formulation was also tested. Two doses of 200 pmol dose of SpCas9-NG and the "CCR5-off1" guide yielded the highest editing rate, with an editing rate of 28.5%. Each condition was compared to a positive control where the formulation was electroporated (without peptide) and to non-treated (NT) as a negative control. Two biological replicates are of cells from two human donors.

[0442] As shown in Figure 20, each peptide mediated delivery of the base editing construct RNP and promoted base editing in T cells to remove the start codon of CCR5 and initiate translation at a frameshifted downstream alternative start codon. The efficiency of base editing can be improved by sequential iterative application of peptides and base editors (delivered by either peptide or electroporation in this example). In this example, PAM mutant enzymes (either "NG" variant or "NRCH" variant) were used.

[0443] Figure 21. Adenine to guanine base editing at the CCR5 locus in primary human T cells delivered by peptide co-incubation with A5K peptide. ABE8e-SpCas9-NG PAM variants were applied to cells with A5K peptide at 20 μM (final concentration). Cells were treated with a single dose, a second dose 2 days after the first dose, or a third dose 2 days after the second dose. Three doses of a 200 pmol dose of SpCas9-NG and the "CCR5-off1" guide yielded the highest editing rate, with an editing rate of 43.6%. Each condition was compared to a positive control where the formulation was electroporated (without peptide) and no treatment as a negative control. Three biological replicates are of cells from three human donors.

[0444] As shown in FIG. 21, each peptide mediated the delivery of the base editing construct RNP (ABE8e-SpCas9-NG) to promote base editing in T cells to remove the start codon of CCR5 and initiate translation at a frameshifted downstream alternative start codon. The efficiency of base editing can be improved by sequential iterative application of peptides and base editors (delivered by either peptide or electroporation in this example). In this example, the "NG" PAM variant enzyme was used. After three consecutive doses of base editor RNP and 20 μM A5K peptide (administrations were given every 2 days), 43.6% base editing was observed at the CCR5 locus.

[0445] FIG. 22. Base editing in primary human HSPCs at the erythroid-specific BCL11a enhancer locus. ABE-8e-NGRNP was applied at a dose of 100 pmol or 200 pmol with 10 μM peptide number 55, and genomic DNA base editing was analyzed by deep sequencing 72 hours after treatment.

[0446] As shown in FIG. 22, peptide no. 55 mediated delivery of the base-edited construct RNP and promoted base editing in HSPCs to convert an “A” nucleotide to a “G” in the erythroid-specific enhancer of Bcl11a.

[0447] Example 4 DNA vaccine We have investigated the use of amphipathic peptides to deliver DNA vaccines (DNA molecules that code for antigenic polypeptides). Figure 23 shows a schematic diagram of peptide-mediated DNA delivery in the context of DNA vaccines, where target cells express the antigens encoded in the delivered DNA, enabling a stable immune response.

[0448] To test delivery to cells in vitro, DNA encoding luciferase was introduced into DC2.4 cells using E5-TAT or E5-R8Q, and the results are shown in Figure 24.

[0449] Figure 24. Luciferase activity showing successful peptide-mediated DNA delivery and protein expression in DC2.4 cells. Luciferase-encoding plasmid was combined with 0, 2, 4, 6, or 8 molar equivalents of dCas9 RNP, which is intended to aid protein expression by binding to the plasmid and delivering DNA into the cell nucleus. 10 μM of peptides E5-TAT or E5-R8Q (peptide number 37) were applied. The combination of peptide E5-R8Q with 4 molar equivalents of RNP resulted in the strongest luciferase expression. No luciferase expression was observed in the absence of peptide or when cells were untreated, and only clear signals were generated when DNA was combined with E5-TAT or E5-R8Q.

[0450] As shown in Figure 24, peptides E5-TAT (Peptide No. 1) and E5-R8Q (Peptide No. 37) mediated plasmid delivery into DC2.4, a cell line that resembles dendritic cells and thus serves as a model for antigen-presenting cells (APCs). In this example, the delivered plasmid contains a luciferase reporter gene and a number of truncated target sequence sites that Cas9 RNP can bind but not cleave. In this example, the most effective delivery was observed when 4 molar equivalents of RNP per plasmid were added and E5-R8Q was used to promote delivery of the macromolecular cargo (RNP-plasmid complex).

[0451] To test in vivo delivery, mice were injected with plasmid DNA encoding the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 in the presence or absence of the E5-TAT peptide. The results are shown in Figure 25.

[0452] Figure 25. Antibody titers measured by ELISA in mice injected with plasmid DNA encoding the RBD of the spike protein from SARS CoV-2 with or without the addition of 200 pmol of E5-TAT peptide. Anti-RBD titers were strongest when 10 μg of plasmid was co-delivered with 200 pmol of E5-TAT.

[0453] As shown in Figure 25, each peptide promoted intracellular delivery of antigen-encoding plasmid DNA, resulting in vaccination of mice against the SARS-CoV-2 spike protein RBD. In this example, no ELISA titers were detected in untreated animals. When antigen-encoding plasmids were administered, there was a high degree of variability between animals in the degree of detectable vaccination (e.g., anti-RBD antibody titers). When the plasmids were administered as part of a formulation containing the peptides, mice consistently showed high anti-RBD antibody titers.

[0454] Example 5 Peptide Screening As shown in Figures 33A-33B, further peptide screening showed that some peptides with sequence elements derived from "parent peptide" numbers 1-37 promoted genome editing in T cells with higher editing efficiency and / or cell viability compared to peptide number 22, one of the best parent peptides. A) Screening of peptide numbers 62-91 (and comparison peptides selected from screening of "parent peptide" numbers 1-61) for Cas9-mediated KO at the CD4 locus in human primary CD4+ T cells, measured by flow cytometry. 50 pmol RNP was applied to cells with each peptide at a concentration of 10 μM. (B) Live / dead fixed violet staining (Thermofisher) and viable cell counts from each treatment condition measured by flow cytometry. Replicate experiments are from two different human cell donors. In both (A) and (B), "DMSO" = Cas9 without peptide, "NT" = cells not treated with Cas9 or peptide. Editing and viability were assessed 3 days after delivery of RNP.

[0455] As shown in Figures 34A-34B, further peptide screening showed that some peptides with sequence elements derived from "parent peptide" nos. 1-91 promoted genome editing in T cells with higher editing efficiency and / or cell viability compared to peptide no. 22, one of the best parent peptides. A) Screening of peptides nos. 92-109 (and comparison peptides selected from the previously screened peptide nos. 1-91 screening) for Cas9-mediated KO at the β2M locus in human primary CD4+ T cells, measured by flow cytometry. 50 pmol of RNP was applied to cells along with peptides at 10 μM concentration. (B) Live / dead fixed cell violet staining (Thermofisher) and viable cell counts from each treatment condition measured by flow cytometry. Replicate experiments are from two different human cell donors. In both (A) and (B), "DMSO" = Cas9 without peptide, "NT" = cells not treated with Cas9 or peptide. Editing and viability were assessed 3 days after delivery of RNP.

[0456] As shown in Figure 35, screening of additional peptides with functional groups (hence "f" in peptides no. f1-15) revealed peptides that retained the efficacy of RNP delivery (and genome editing) in primary human T cells, some of which supported higher editing efficiency and / or cell viability compared to peptide no. 22, one of the best parent peptides. Left: Screening of peptides f1 / f2 / f3 / f5 / f6 / f11 (and comparison peptides selected from previous screening) for Cas9-mediated B2M KO in human primary CD4+ T cells, measured by flow cytometry. 50 pmol of RNP was applied to cells together with peptides at 10 μM concentration. Right: Number of live cells from each treatment condition measured by live / dead fixed violet staining (Thermofisher) and flow cytometry. Replicate experiments are from two different human cell donors. Editing and viability were evaluated 3 days after delivery of RNP.

[0457] As shown in Figure 36, further peptide screening showed that some (but not all) peptides with sequence elements derived from the "parent peptides" nos. 1-37 promoted genome editing in HSPCs with higher editing efficiency and / or cell viability compared to peptide no. 55, one of the peptides that performed best in these respects in HSPCs in the previous round of screening. In this screen, Cas9 RNP targeted β2M for KO and editing was detected by flow cytometry. Top left: Human primary CD34 cells measured by flow cytometry 3.5 days after treatment with 50 pmol of RNP and 5 μM or 10 μM concentrations of peptide (medium grey and black bars, respectively). + Screening of peptides 61-91 for Cas9-mediated KO at the β2M locus in hematopoietic stem and progenitor cells (HSPCs) (shown alongside comparative peptides 31 and 55 from a previous screen). Top right: Number of β2M edited cells detected by flow cytometry 3.5 days after delivery plotted for each peptide screened. Bottom left: Viability assessed by Cell Titre Glo (CTG) assay 24 hours after treatment with RNP and peptide. "DMSO" = Cas9 without peptide, "solvent only" = cells not treated with Cas9 or peptide. "No peptide" data points are labeled "NP" and shown in light grey.

[0458] As shown in Figure 37, further peptide screening showed that some (but not all) peptides with sequence elements derived from "parent peptide" numbers 1-37 promoted genome editing in HSPCs with higher editing efficiency and / or cell viability compared to peptide number 55, one of the peptides that showed the highest performance in these respects in HSPCs in the previous round of screening. In this screen, Cas9 RNP targeted β2M for KO and editing was detected by flow cytometry. Top: Human primary CD34 cells measured by flow cytometry 3.5 days after treatment with 50 pmol RNP and peptide at 5 μM or 10 μM concentration. + Screening of peptides 92-109 for Cas9-mediated KO at the β2M locus in hematopoietic stem and progenitor cells (HSPCs) (shown alongside some comparative peptides from a previous screen). Bottom: Viability assessed by Cell Titre Glo (CTG) assay 24 hours after treatment with RNP and peptides. "DMSO" = Cas9 RNP without peptide, "NT" = cells not treated with Cas9 or peptide. Colored data points and bars do not reflect peptide concentrations in "DMSO" and "NT" as no peptide was added to the cells.

[0459] Example 6 Gene Editing As shown in Figure 38, screening of further peptides described in Example 5 showed that some (but not all) of the peptides with sequence elements extracted from the "parent peptides" nos. 1-37 promoted genome editing in neural progenitor cells (NPCs) derived from Ai9 mice with higher editing efficiency and / or cell viability compared to peptides nos. 22 and 55, two of the peptides that showed the highest performance in these respects in Ai9-derived NPCs in previous rounds of screening. Screening of peptides nos. 61-91 for Cas9-mediated cleavage of the tdTomato repressor in Ai9 NPCs was performed in parallel with comparative peptides nos. 55 and 22 (A5K). Editing efficiency measured by red fluorescent protein expression by flow cytometry 3 days after treatment with 50 pmol of RNP and peptide at a concentration of 10 μM.

[0460] As shown in Figures 39A-39D, peptide-mediated delivery of Cas9 RNP can promote the generation of CAR-T cells capable of effective tumor killing in an in vivo xenograft mouse model with comparable potency to cells generated using electroporation. a. Schematic of in vivo tumor challenge experiment with n=8 mice per group. b. NALM6 cytotoxicity assay. Error bars represent SEM from three technical replicates. c. Bioluminescence imaging values ​​at the last measurement day when all mice injected with CAR-T cells were alive. p-values ​​are derived from unpaired two-tailed Welch t-test. d. Kaplan-Meier survival analysis. p-values ​​are derived from log-rank test. P<0.001 for each comparison of edited conditions vs. nt cells.

[0461] As shown in Figures 40A-40D, peptide-mediated delivery of Cas9RNP can facilitate the generation of CAR-T cells capable of effective tumor killing in ex vivo assays with potency comparable to cells generated using electroporation and superior to CAR-T cells generated using gammaretrovirus for CAR delivery. a. Schematic of repeated stimulation and cytotoxicity assay. CD3+ T cells were edited to express CAR using either gRV, Cas12a RNP electroporation and AAV, or Cas12a RNP PERC and AAV. b. Percentage of CAR+ cells in each condition in the presence or absence of repeated stimulation using CD19+ A549 cells. c., d. NALM6 cytotoxicity assays and area under curve analysis in T cells in each condition. n=3 biological replicates from different human donors. Bars represent the mean. Error bars represent SEM (3 biological replicates x 3 technical replicates). p-values ​​are derived from two-tailed Welch's unpaired t-test.

[0462] As shown in Figure 41, peptide-mediated delivery of Cas9 RNP is effective when using a variety of different Cas9 protein constructs of academic or commercial origin. Figure 41 shows a comparison of editing efficiency and survival rate after peptide-mediated delivery with peptide 22 (A5K, "PERC") or electroporation (e-por) of various S. pyogenes Cas9 protein constructs. Two published NLS-rich constructs were purified in the lab: triNLS (similar to "3xNLS" in Wu et al. doi:10.1038 / s41591-019-0401-y) and 6xNLS (similar to "4xNLS" in Staahl et al. (2017) Nat. Biotechnol. 35:431). The other four constructs were from commercial vendors (TrueCut-v2: Invitrogen TrueCut Cas9 Protein v2 (ThermoFisher), Synthego: SpCas9 2NLS (Synthego), IDT-v3: Alt-R SpCas9 Nuclease V3 (IDT), IDT-v3 HiFi: Alt-R SpHiFi Cas9 Nuclease V3 (IDT)).

[0463] As shown in Figures 42A-42D, peptide-mediated delivery ("PERC") helps maintain cell viability and phenotype during sequential editing. + a. Schematic of sequential editing of three loci in T cells. b. Comparison of editing using PERC (peptide no. 22, A5K) with electroporation as measured by flow cytometry for TCR, CAR, β2M, and CD5 surface expression. Reported CAR+ cells are TCR - c. Editing without CAR AAV. The number of cells in each condition was 4 × 10 6Scaled for T cells. Bars represent only cells with all edits attempted, not cells with only partial edits attempted. n=3 biological replicates from different human donors. Bars represent mean. Error bars represent SEM. p values ​​are from unpaired two-tailed Welch t-test. D. CD4 expression between each delivery method measured by flow cytometry for surface expression of CD62L and CD45RA. + and CD8 + Comparison of cells (irrelevant to the outcome of editing). Pie charts represent the percentage of different cell phenotypes observed after three consecutive rounds of PERC (middle pair) or electroporation (bottom pair) compared to non-treated cells (top pair). Each segment of the pie chart represents the average percentage of each phenotype. Dotted lines indicate comparisons, p-values ​​were obtained from two-way ANOVA and Holm-Sidak multiple comparison test.

[0464] As shown in Figures 43A-43D, peptide-mediated delivery ("PERC") aids cell viability in the generation of engineered T cells with multiple knock-in edits at different genomic loci. This can be done using only Cas9 RNPs or a mixture of Cas9 RNPs and Cas12a RNPs (one per genomic locus). a., b. CD8 T cells with Cas9 and PERC (Peptide No. 22 / A5K) + Sequential and simultaneous double knock-in editing by PERC (peptide no. 22 / A5K) or e-por (electroporation) in T cells. c, d. CD3 + Sequential and simultaneous double knock-in editing by PERC (peptide no. 22 / A5K) or e-por (electroporation) in T cells. Bars represent cells in which both knock-in edits were attempted. Flow cytometry was performed 6 days after the first edit. n=3 biological replicates from different human donors. Bars represent the mean. Error bars represent SEM. p-values ​​are derived from unpaired two-tailed Welch t-test.

[0465] As shown in Figures 44A-44F, peptide-mediated delivery of CRISPR RNPs ("PERC") aids in the modification of T cells while minimizing induced T cell phenotypic perturbations, especially compared to electroporation of RNPs. a. Volcano plots show fold changes in gene expression and adjusted p-values ​​incorporating data from 6 hours, 1 day, and 7 days after editing by RNP delivery using either peptide number 22 (PERC) or electroporation (e-por). In the "DMSO" condition, cells are exposed to RNPs and 0.1% DMSO (the same amount that would result from delivery of the peptide dissolved in DMSO), but no peptide. This plot combines data from all three time points. Set of 84 genes that were significantly downregulated (b) or upregulated (c) in one or more conditions in b–ea, d results for each condition (asterisk indicates one gene significantly affected by PERC / DMSO), annotations and gene labels for NanoString gene categories in e / c / d, fold change between gene categories using genes and annotations in f / c / d / e. Dots represent individual genes and shaded curves represent distribution of fold change.

[0466] As shown in Figure 45, peptide-mediated delivery of CRISPR RNPs ("PERC") aids in multiplex genome editing while minimizing the induction of chromosomal translocations, especially when compared to simultaneous electroporation of RNP nucleases targeting multiple genomic loci. The figure shows the analysis of translocation frequency by ddPCR upon sequential or simultaneous treatment with one or two Cas9 nuclease RNPs (targeting either TRAC or β2M). Editing is consistent with the CD3 + This was performed using PERC (peptide no. 22 / A5K) or e-por (electroporation) in T cells. Each experiment included n=2-3 biological replicates from different human donors. Bars represent the mean. Error bars represent SEM. p-values ​​are derived from unpaired two-tailed Welch t-test.

[0467] As shown in Figures 46A-46C, peptide-mediated delivery of CRISPR RNPs ("PERC") aids in improved cell yield and stable cell growth over time after T cell engineering, especially when compared to electroporation of RNPs. a. Depiction of metrics to assess improved cell generation. b. Schematic of sequential editing. c. Growth of edited cells over time versus number of cells used for editing. n=3 biological replicates are from different human donors and are plotted separately. Editing enhances CD3 + This was performed in T cells by PERC (peptide no. 22 / A5K) or e-por (electroporation).

[0468] As shown in Figure 47, peptide-mediated delivery of CRISPR RNPs ("PERC") aids in the precise knock-in of genes into primary human B cells when AAV6 is used to provide the necessary DNA donor template to enable homologous recombination repair (HDR). The figure shows primary human B cell knock-in using Cas9 RNPs delivered via electroporation (epor) or the A5K peptide (A5K, peptide number 22) and HDR template delivered via AAV6 (KI) to create an sfGFP fusion to the N-terminal side of clathrin by targeting CLTA exon 1 (green) or as an off-target control at the β2M locus (blue), with no AAV6 condition (KO) and untreated cells (NT) shown for comparison. GFP expression was measured by flow cytometry at day 7 and shown as the percentage of live cells expressing GFP (left) and the number of live cells expressing GFP (right).

[0469] As shown in Figure 48, peptide-mediated delivery of CRISPR RNPs ("PERC") aids in the precise knock-in of genes into primary human NK cells when AAV6 is used to provide the necessary DNA donor template to enable HDR. The figure shows primary human NK cell knock-in of sfGFP fusions to the N-terminal side of clathrin by targeting CLTA exon 1 using peptide #22 / A5K (A5K KO) or a Cas9 RNP delivered by electroporation (Epor) and HDR template delivered by AAV6 (+AAV), with AAV-free scene (KO) and non-treated cell (NT) conditions shown for comparison. GFP expression was measured by flow cytometry on day 7 and shown as the percentage of live cells expressing GFP (left) and the number of live cells expressing GFP (right).

[0470] As shown in Figure 49, peptide-mediated delivery of CRISPR Cas9 RNP using a functionally identical gRNA to that shown to confer clinical benefit in hemoglobinopathies following use in ex vivo editing and transplantation of CRISPR-edited HSPCs resulted in primary human CD34+ / -1 (CD34+ / -1) expression at the BCL11a locus. + This aids in highly efficient genome editing of HSPCs (Frangoul et al. (2021) N Engl J Med 384:252). This figure shows the results of cultured primary human CD34 +Results are shown after treating HSPCs with various amounts of peptide number 55 and various amounts of RNP containing Cas9 protein and gRNA with the spacer sequence "1617" reported by Wu et al. Nat. Med. (2019) 25:5 and used in Frangoul et al. (2021) supra. The graph on the left shows the editing rate detected by amplicon-based NGS of genomic DNA harvested 72 hours after peptide-mediated delivery of Cas9 RNP containing "1617" gRNA targeting BCL11a. The graph on the right shows the viability 24 hours after the same delivery event shown in the graph on the left. 50, 100, or 200 pmol of RNP was delivered in combination with 0, 5, or 10 μM of peptide (measured based on the final concentration in the cell medium). Two technical replicates were performed in the 50 pmol and 100 pmol conditions and one technical replicate was performed in the 200 pmol condition. The experiment was performed using 20,000 cells in 100 μL of medium.

[0471] As shown in Figure 50, peptide-mediated delivery of CRISPR Cas9 RNP inhibits the expression of primary human CD34 + Supports highly efficient genome editing at the B2M locus in HSPCs. This figure shows cultured primary human CD34 + Results are shown after treating HSPCs with varying amounts of peptide number 55 and varying amounts of Cas9 RNP. The graph on the left shows the editing rate detected by amplicon-based NGS of genomic DNA harvested 6 days after peptide-mediated delivery of Cas9 RNP containing a gRNA targeting B2M. The graph on the right shows the viability 24 hours after the same delivery event shown in the graph on the left. 100 pmol of RNP was delivered with 10 μM peptide (measured based on the final concentration in cell media). Two technical replicates were performed. The experiment was performed using 100,000 cells in 100 μL of media.

[0472] Example 7. Base editing As shown in Figure 51, base editing of primary human HSPCs at the erythroid-specific BCL11a enhancer locus, a dose of 50 pmol of ABE-8e-NGG RNP was applied along with 10 μM of peptide number 55, and genomic DNA base editing was analyzed by deep sequencing 72 hours after treatment. This experiment uses the gRNA sequence reported in Richter et.al Nat. Biotech. (2020) 38:7, which can induce an adenine base editor to destroy the erythroid-specific enhancer of BCL11a, a protein that suppresses the expression of fetal hemoglobin.

[0473] Example 8. In vivo gene editing The use of peptides to promote genome editing in vivo was examined, and the results are shown in Figure 52, Figures 53A-53B, and Figure 54.

[0474] As shown in Figure 52, formulations containing Cas9 RNP (loaded with equal amounts of two gRNAs capable of inducing the excision of the repressor of tdTomato RFP) and peptide number 55 can promote genome editing in striatal neurons in mouse brains. 7 μL of each RNP formulation was injected into the striatum of Ai9 mouse brains using convection-enhanced delivery (CED). The left striatum was injected with the RNP-tween formulation, and the right striatum was injected with the RNP-peptide-tween formulation (containing peptide 55). The reported amounts of peptide (2 mM) and Tween-80 (0.05%) refer to the concentrations in the 7 μL formulation injected. In the 7 μL formulation injected, the RNP concentration was 54.4 μM in the sample containing peptide 55 and 52.6 μM in the sample without peptide 55. Tissue sections were stained for NeuN (neurons), DAPI (nuclei), and tdTomato (polyclonal rabbit anti-RFP from Invitrogen). Images are adjusted to maximize the red signal from tdTomato. n=2-3 mice.

[0475] Figures 53A-53B report quantification of neuronal editing in Ai9 mice, where (a) represents the editing rate detected in an RNP / tween-containing formulation that also contains peptide number 55, and (b) represents the editing rate detected in an RNP / tween-containing formulation without the peptide.

[0476] As shown in Figure 54, an intravenous formulation containing Cas9 RNP and peptides can promote genome editing of human primary T cells in vivo for a humanized mouse model (NSG mice injected with human peripheral blood mononuclear cells). Editing efficiency was assessed by NGS in splenocytes from humanized mice administered either Cas9RNP targeting the β2M locus and peptide number 1 (E5-TAT), or peptide number 22 (A5K) combined with a CD3 targeting antibody. Splenocytes were further sorted by FACS into T cell and non-T cell populations, and indels were detected in the populations by NGS. Editing rates were increased when Cas9RNP was non-covalently linked (via a protein A fusion construct of Cas9) to the anti-CD3 antibody OKT3 (targeting the human T cell receptor), which was used in an "Fc silencing" format that prevents binding by Fc receptor proteins.

[0477] Although the present invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

1. (1) Cargo, and (2) A cargo-delivery fusion polypeptide comprising: a) an endosomolytic polypeptide; and b) a cell-penetrating polypeptide. A composition comprising: The cargo is (i) a CRISPR-Cas effector polypeptide, or a CRISPR-Cas fusion polypeptide comprising a CRISPR-Cas effector polypeptide and one or more heterologous polypeptides; and / or (ii) a DNA molecule (iia) encoding a polypeptide, and / or (iib) having a length of 100 nucleotides or more and containing sufficient sequence to support homology-directed repair. Including, the cargo delivery fusion polypeptide comprises the amino acid sequence of any one of formulas I to X: the cargo delivery fusion polypeptide has a length of about 32 amino acids to about 35 amino acids; and any two adjacent amino acids are independently linked by an amide bond or a non-amide bond; Formula I is KLFEX １ IEGFIENGWEX ２ MIDX ３ WX ４ GX ５ GRKKRRQRR (SEQ ID NO: 165), wherein: X １ is A, R, or K; X ２ is A or G, X ３ is L or G, X ４ is N or Y, X ５ is Y, if present; Formula II is X １ LFEX ２ IEGFIENGWEGMIDGWYGYGRKKRRQRR (SEQ ID NO: 166), wherein X １ is R or G, and X ２ is R or K; Formula III is GLFEAIEGFIENGWEX １ MIDX ２ WNGYGRKKRRQRR (SEQ ID NO: 167), wherein X １ is A or G, and X ２ is G or L; Formula IV is GLFEAIEGFIENGWEX １ X ２ IX ３ LWYGYGRKKRRQRR (SEQ ID NO: 168), wherein: X １ is A or G, X ２ is L or M, X ３ is D or E; Formula V is GLFX １ AIAX ２ FIX ３ NGWX ４ GLIX ５ GWYGGRKKRRQRRR (SEQ ID NO: 208), wherein X １ , X ２ , X ３ , X ４ , and X ５ each of which is independently a non-encoded amino acid; Formula VI is KLFEX １ IX ２ X ３ FIENGWEGMIX ４ X ５ WX ６ GYGRKKRRQRX ７ (SEQ ID NO: 170), wherein X １ is A or H, and X ２ is E or A, and X ３ is G or E, and X ４ is D or E, and X ５ is G or L, and X ６ is E, H, K, R, or N, and X ７ is, if present, R; Formula VII is GLFEX １ IX ２ X ３ FIENGWEGMIDX ４ WX ５ GYGRKKRRQRR (SEQ ID NO: 171), wherein X １ is R, H, A, or K, and X ２ is E or A, and X ３ is G or E, and X ４ is L or G, and X ５ is N, Y, K, or E; Formula VIII is HLFEX １ IEGFIENGWEGMIDX ２ WX ３ GYGRKKRRQRR (SEQ ID NO: 172), wherein X １ is A or K, and X ２ is G or L, and X ３ is N, K, E, or Y; Formula IX is KLFEAIEGFIENGWEGMIDLWNX １ X ２ YGRKKRRQRR (SEQ ID NO: 173), wherein X １ is, if present, Gly; X ２ is Cys(methyltetrazine) or Cys(3-nitro-2-pyridinesulfenyl); Formula X is KLFEAIEGFIENGWEGMIDLWNGX １ YGRKKRRQRRX ２ (SEQ ID NO: 174), wherein X １ Cys(methyltetrazine-PEG4-maleimide), Cys(maleimide), Lys(PEG ２３ ) ２ , Lys(3-nitro-pyridine-2-carboxylic acid), Lys(PEG ２３ ) ２ , Lys(PEG ２３ ) ２ -(3-nitro-pyridine-2-carboxylic acid), or Cys(1,4-bis(bromomethyl)-benzene), and X ２ is Cys(3-nitro-2-pyridine-sulfenyl) or Lys(methyltetrazine-PEG4).

2. (A) the cargo delivery fusion polypeptide comprises: (i) KLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 19, SEQ ID NO: 19); KLFEAIEGFIENGWEGMIDGWYG GRKKRRQRR (Peptide 40, SEQ ID NO: 40), KLFERIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 44, SEQ ID NO: 44), KLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 45, SEQ ID NO: 45), KLFEAIEGFIENGWEAMIDGWYGYGRKKRRQRR (Peptide 46, SEQ ID NO: 46), KLFEAIEGFIENGWEGMIDLWYGYGRKKRRQRR (Peptide 47, SEQ ID NO: 47), KLFEAIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 48, SEQ ID NO: 48), KLFEAIEGFIENGWEAMIDLWYGYGRKKRRQRR (Peptide 53, SEQ ID NO: 53), KLFEAIEGFIENGWEAMIDGWNGYGRKKRRQRR (Peptide 54, SEQ ID NO: 54), KLFEAIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 55, SEQ ID NO: 55), KLFEAIEGFIENGWEAMIDLWNGYGRKKRRQRR (Peptide 56, SEQ ID NO: 56), and KLFEKIEGFIENGWEAMIDLWNGYGRKKRRQRR (Peptide 57, SEQ ID NO: 57) an amino acid sequence of Formula I selected from the group consisting of: or (ii) RLFERIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 42, SEQ ID NO: 42); RLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 43, SEQ ID NO: 43), and GLFERIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 41, SEQ ID NO: 41) an amino acid sequence of Formula II selected from the group consisting of: or (iii) GLFEAIEGFIENGWEAMIDGWNGYGRKKRRQRR (Peptide 50, SEQ ID NO: 50); GLFEAIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 51, SEQ ID NO: 51), and GLFEAIEGFIENGWEAMIDLWNGYGRKKRRQRR (Peptide 52, SEQ ID NO: 52) an amino acid sequence of Formula III selected from the group consisting of: or (iv) GLFEAIEGFIENGWEAMIDLWYGYGRKKRRQRR (Peptide 49, SEQ ID NO: 49), and GLFEAIEGFIENGWEGLIELWYGYGRKKRRQRR (Peptide 58, SEQ ID NO: 58) an amino acid sequence of Formula IV selected from: or (v) GLFαAIAαFIαNGWαGLIαGWYGGRKKRRQRRR (Peptide 59, SEQ ID NO: 59), or GLFαAIAαFIENGWEGLIDGWYGGRKKRRQRRR (Peptide 60, SEQ ID NO: 60) and / or an amino acid sequence of formula V selected from the group consisting of １ , X ２ , X ３ , X ４ , and X ５ is α-aminoadipic acid; or (vi) KLFEAIEGFIENGWEGMIDLWEGYGRKKRRQRR (Peptide 62, SEQ ID NO: 62); KLFEAIEGFIENGWEGMIDLWHGYGRKKRRQRR (Peptide 63, SEQ ID NO: 63), KLFEAIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 64, SEQ ID NO: 64), KLFEAIEGFIENGWEGMIDLWRGYGRKKRRQRR (Peptide 65, SEQ ID NO: 65), KLFEAIEGFIENGWEGMIDLWNGYGRKKRRQR (Peptide 69, SEQ ID NO: 69), KLFEAIEGFIENGWEGMIELWNGYGRKKRRQRR (Peptide 71, SEQ ID NO: 71), KLFEAIAEFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 72, SEQ ID NO: 72), KLFEHIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 105, SEQ ID NO: 105), KLFEHIEGFIENGWEGMIDLWYGYGRKKRRQRR (Peptide 107, SEQ ID NO: 107), and KLFEHIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 109, SEQ ID NO: 109) an amino acid sequence of Formula VI selected from: or (vii) GLFERIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 66, SEQ ID NO: 66); GLFEHIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 68, SEQ ID NO: 68), GLFEAIAEFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 70, SEQ ID NO: 70), GLFEKIEGFIENGWEGMIDLWYGYGRKKRRQRR (Peptide 74, SEQ ID NO: 74), GLFEAIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 75, SEQ ID NO: 31), GLFEAIEEFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 76, SEQ ID NO: 24), GLFEKIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 98, SEQ ID NO: 98), GLFEKIEGFIENGWEGMIDGWKGYGRKKRRQRR (Peptide 99, SEQ ID NO: 99), GLFEKIEGFIENGWEGMIDGWEGYGRKKRRQRR (Peptide 100, SEQ ID NO: 100), GLFEKIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 101, SEQ ID NO: 101), GLFEKIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 102, SEQ ID NO: 102), and GLFEKIEGFIENGWEGMIDLWEGYGRKKRRQRR (Peptide 103, SEQ ID NO: 103) an amino acid sequence of Formula VII selected from: or (viii) HLFEAIEGFIENGWEGMIDGWNGYGRKKRRQRR (Peptide 92, SEQ ID NO: 92); HLFEAIEGFIENGWEGMIDGWKGYGRKKRRQRR (Peptide 93, SEQ ID NO: 93), HLFEAIEGFIENGWEGMIDGWEGYGRKKRRQRR (Peptide 94, SEQ ID NO: 94), HLFEAIEGFIENGWEGMIDLWNGYGRKKRRQRR (Peptide 95, SEQ ID NO: 67), HLFEAIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 96, SEQ ID NO: 96), HLFEAIEGFIENGWEGMIDLWEGYGRKKRRQRR (Peptide 97, SEQ ID NO: 97), HLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRR (Peptide 104, SEQ ID NO: 104), HLFEKIEGFIENGWEGMIDLWYGYGRKKRRQRR (Peptide 106, SEQ ID NO: 106), and HLFEKIEGFIENGWEGMIDLWKGYGRKKRRQRR (Peptide 108, SEQ ID NO: 108) an amino acid sequence of Formula VIII selected from: or (ix)KLFEAIEGFIENGWEGMIDLWNX １ X ２ YGRKKRRQRR (SEQ ID NO: 173) １ is, if present, Gly; X ２ is Cys(methyltetrazine) or Cys(3-nitro-2-pyridinesulfenyl)), KLFEAIEGFIENGWEGMIDLWNC*YGRKKRRQRR (Peptide 87, SEQ ID NO: 87) (where "C*" is Cys (methyltetrazine)); KLFEAIEGFIENGWEGMIDLWNGC*YGRKKRRQRR (Peptide 88, SEQ ID NO: 88) (where "C*" is Cys (methyltetrazine)); KLFEAIEGFIENGWEGMIDLWNC*YGRKKRRQRR (Peptide 89, SEQ ID NO:89) (where "C*" is Cys(3-nitro-2-pyridinesulfenyl)), and KLFEAIEGFIENGWEGMIDLWNGC*YGRKKRRQRR (Peptide 90, SEQ ID NO: 90) (where "C*" is Cys(3-nitro-2-pyridinesulfenyl)) an amino acid sequence of Formula IX selected from: or (x)KLFEAIEGFIENGWEGMIDLWNGX １ YGRKKRRQRRX ２ (SEQ ID NO: 174) (wherein X １ Cys(methyltetrazine-PEG4-maleimide), Cys(maleimide), Lys(PEG ２３ ) ２ , Lys(3-nitro-pyridine-2-carboxylic acid), Lys(PEG ２３ ) ２ , Lys(PEG ２３ ) ２ -(3-nitro-pyridine-2-carboxylic acid), or Cys(1,4-bis(bromomethyl)-benzene), and X ２ is Cys(3-nitro-2-pyridine-sulfenyl) or Lys(methyltetrazine-PEG4)), The amino acid sequence of peptide f1, the amino acid sequence of peptide f2, the amino acid sequence of peptide f3, the amino acid sequence of peptide f4, the amino acid sequence of peptide f5, the amino acid sequence of peptide f6, the amino acid sequence of peptide f7, the amino acid sequence of peptide f11, the amino acid sequence of peptide f13, and the amino acid sequence of peptide f14 shown in FIG. an amino acid sequence of formula X selected from Including, and / or (B) all of the amino acids in the cargo delivery fusion polypeptide are linked by amide bonds, or at least two adjacent amino acids are linked by a non-amide bond; The composition of claim 1.

3. (A) one or more of the amino acids in the polypeptide comprises a modification, and optionally (i) the modification comprises one or more of the following: a maleimide group, a methyltetrazine group, a 3-nitro-pyridine-2-carboxylic acid group, a 1,4-bis(bromomethyl)-benzene group, a poly(ethylene glycol) group, a 5-carboxyfluorescein group, a nitropyridine group, a pyridyl disulfide, and a pyridine; and / or (B) at least two adjacent amino acids are linked by a linker comprising one or more ethylene glycol monomers, and optionally the linker is a polymer comprising 2, 4, 6, or 8 ethylene glycol monomers; The composition of claim 1.

4. the cargo delivery fusion polypeptide (i) comprising the amino acid sequence of any one of peptides 19 and 40-60 shown in Figure 1; or (ii) comprises the amino acid sequence of any one of the peptides shown in Figure 30; or (ii) does not contain the amino acid sequence of any of the peptides designated 1-18 or 21-27 in Figure 1; The composition of claim 1.

5. The composition of any one of claims 1 to 4, wherein the cargo comprises the DNA molecule.

6. The composition of any one of claims 1 to 4, wherein the cargo comprises the CRISPR-Cas effector polypeptide or the CRISPR-Cas fusion polypeptide.

7. 7. The composition of claim 6, comprising a CRISPR-Cas guide nucleic acid.

8. 7. The composition of claim 6, comprising a donor DNA template and / or wherein the CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide, a type V CRISPR-Cas effector polypeptide, or a type VI CRISPR-Cas effector polypeptide.

9. 8. The composition of claim 7, wherein the CRISPR-Cas guide nucleic acid is RNA, optionally wherein the CRISPR-Cas guide nucleic acid is a unimolecular guide RNA or a bimolecular guide RNA.

10. (A) the composition comprises: (i) a CRISPR-Cas effector polypeptide; and (ii) one or more nuclear localization signals; or one or more heterologous effector polypeptides, optionally wherein at least one of the one or more heterologous effector polypeptides is a single-stranded nuclease, a double-stranded nuclease, a helicase, a methylase, a demethylase, an acetylase, a deacetylase, a deaminase, an integrase, a recombinase, a base editor, or a prime editor. a CRISPR-Cas fusion polypeptide comprising: and / or (B) the CRISPR-Cas effector polypeptide or the CRISPR-Cas fusion polypeptide is (i) a covalently linked targeting moiety, optionally an antibody, including Protein A, Protein G, an aptamer, a DARPin, or an antibody optionally non-covalently linked to an affinity moiety, and / or optionally, the antibody specifically binds to an epitope on the surface of a eukaryotic cell, thereby targeting the composition to the cell; and / or (ii) a non-polypeptide polymer, which is optionally poly(ethylene glycol). Including, The composition of claim 6.

11. 8. The composition of claim 7, wherein the CRISPR-Cas effector polypeptide and the guide nucleic acid are present in a ribonucleoprotein (RNP) complex, optionally at a molar ratio of cargo delivery fusion polypeptide to RNP of at least 3:1, optionally at a molar ratio of cargo delivery fusion polypeptide to RNP of 10:1 to 50:

1.

12. (i) the cargo delivery fusion polypeptide is present in the composition at a concentration of about 2 μM to about 50 μM; and / or (ii) comprising one or more of a solubilizer, a surfactant, a buffer, a salt, and a protease inhibitor; and / or (iii) comprising poly(ethylene glycol), a non-ionic surfactant, or both; The composition of claim 1.

13. 10. A method of delivering a cargo to a target population of eukaryotic cells, comprising contacting said cells with the composition of claim 1, thereby producing an altered target population of eukaryotic cells comprising said cargo, and optionally: (A) the target population of eukaryotic cells comprises T cells, stem cells, natural killer cells, kidney cells, or neuronal cells; or the target eukaryotic cells are hematopoietic stem cells or hematopoietic progenitor cells; and / or (B) (i) the cell is in vitro; optionally (a) after said contacting step, at least 50% of said target population of eukaryotic cells retain viability; and / or (b) introducing a second composition comprising a second cargo into said modified target population of eukaryotic cells, optionally wherein said introducing is by electroporation or transfection, and optionally wherein said transfection comprises contacting said modified target population of eukaryotic cells with a recombinant viral vector; or (ii) the cells are ex vivo; The method.

14. 1. A method for delivering a cargo, which is a DNA molecule or a ribonucleoprotein (RNP), to a eukaryotic cell, comprising contacting the cell with a composition comprising: (I) and (II): (I) an amphipathic cargo-delivery fusion polypeptide, (i) an endosomolytic polypeptide; and (ii) a cell-penetrating polypeptide; the amphipathic cargo delivery fusion polypeptide comprising: and (II) (A) if the cargo is a DNA molecule, (Ai) a DNA molecule encoding a polypeptide, and / or (Aii) a DNA molecule having a length of 100 nucleotides or more and containing sufficient sequences to support homology-directed repair; optionally, (i) the immunogenic polypeptide is a viral polypeptide; and / or (ii) the endosomolytic polypeptide is a) comprises the amino acid sequence GLFEAIAEFIENGWEGLIEGWYG (SEQ ID NO: 163); b) contains 1 to 5 amino acid substitutions relative to GLFEAIAEFIENGWEGLIEGWYG (SEQ ID NO: 163); c) comprising the amino acid sequence GLFEAIEGFIENGWEGMIDGWYG (SEQ ID NO: 164); or d) contains 1 to 5 amino acid substitutions relative to GLFEAIEGFIENGWEGMIDGWYG (SEQ ID NO: 164); the endosomolytic polypeptide has a length of about 20 amino acids to about 30 amino acids; and / or (iii) the cell-penetrating polypeptide comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 207), YGRKKRRQRR (SEQ ID NO: 160), or GRKKRRQRRR (SEQ ID NO: 161), and has a length of 10 to 15 amino acids; or (B) when the cargo is a ribonucleoprotein (RNP), i) a CRISPR-Cas effector polypeptide; and ii) a guide nucleic acid; RNPs, optionally comprising: (i) the endosomolytic polypeptide is a) comprising the amino acid sequence GLFEAIAEFIENGWEGLIEGWYG (SEQ ID NO: 163); or b) contains 1 to 5 amino acid substitutions relative to GLFEAIAEFIENGWEGLIEGWYG (SEQ ID NO: 163); or c) comprising the amino acid sequence GLFEAIEGFIENGWEGMIDGWYG (SEQ ID NO: 164); or d) contains 1 to 5 amino acid substitutions relative to GLFEAIEGFIENGWEGMIDGWYG (SEQ ID NO: 164); the endosomolytic polypeptide has a length of about 20 amino acids to about 30 amino acids; and / or (ii) the cell-penetrating polypeptide comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 207), YGRKKRRQRR (SEQ ID NO: 160), or GRKKRRQRRR (SEQ ID NO: 161), and has a length of 10 to 15 amino acids; and / or (iii) the method comprises introducing a DNA donor template into the cell; and optionally (a) the donor template is present within a recombinant viral vector, optionally wherein the recombinant viral vector is a recombinant adeno-associated viral vector; and / or (b) the donor template comprises a nucleotide sequence encoding a polypeptide, optionally wherein the polypeptide is a chimeric antigen receptor comprising a single-chain Fv or nanobody specific for a cancer-associated antigen; and / or (iv) the eukaryotic cell is an immune cell, optionally wherein the immune cell is a T cell, a B cell, or an NK cell; and / or (v) the eukaryotic cell is (a) ex vivo, or (b) in vitro.

15. A composition according to any one of claims 1 to 4 for use as a medicament.

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