WO2021118927A1 - Methods and compositions for targeted delivery of nucleic acid therapeutics - Google Patents

Abstract

Methods and compositions for the specific delivery of nucleic acid encoded therapeutics using targetmers. Targetmers comprise a minor groove binder linked to a ligand for a receptor that is expressed on the target cell.

Description

METHODS AND COMPOSITIONS FOR TARGETED DELIVERY OF NUCLEIC ACID THERAPEUTICS RELATED APPLICATIONS [0001] This application claims the benefit under 35 USC 119(e) of U.S. Provisional Application No. 62/947,563, filed on December 13, 2019, which is incorporated herein by reference in its entirety. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 4, 2020, is named 0371_0008WO1_SL.txt and is 1,825 bytes in size. BACKGROUND OF THE INVENTION [0003] Various methods have been developed to deliver therapeutic nucleic acids to tissues not limited to viruses, nanoparticles or conjugation to other molecules. Targeted delivery to liver is successful for N-acetylgalactosamine (GalNAc) conjugates that bind with high affinity to the asialoglycoprotein receptor (ASGPR) [1]. Also approaches on conjugating aptamers that target specific receptors also report successful targeting of small nucleic acid to surfaces that bear appropriate receptors [2, 3]. A number of different nucleic chemistries allow optimization of these therapeutics for treatment of disease in humans [4]. Larger nucleic acids are often encapsulated in viral proteins [5, 6], nanoparticles [7] and by physical means [8]. Still targeted gene delivery remains a problem in the clinic [9]. Manufacture of viruses is challenging, as are the immune responses against foreign proteins that limit redosing. Targeting with nano-particles is limited through uptake by scavenger cells in different tissues limiting their selectivity, difficulties in manufacture and potential toxicities [10]. [0004] There are reports of other approaches using “naked” plasmid DNA, but the efficiency of delivery is low. Some unmodified RNAs are taken up through a process called gymnosis [11], but this method is not targeted. Noncovalent binding off small RNAs to a poly-arginine tail attached to an antibody has been one approach to improve delivery to a specific cell type [12]. Another approach is to deliver a therapeutic directly to the cytoplasm using a membrane binding steroid linked to ethidium bromide that intercalates into any double-stranded region of a nucleic acid with only limited sequence specificity [13]. While this approach solves the delivery problem, the therapeutic is not targeted to any specific cell type.

[ 0005 ] Molecules that bind to the minor groove of a double-stranded nucleic acid structure offer a new way to attach targeting ligands to a nucleic acid therapeutic. They have many properties that are useful for the design of a delivery strategy for nucleic acid therapeutics.

[ 0006] Minor grove binders are specific for short sequence motifs of 4 or more bases allowing the number that bind the therapeutic to be determined from its sequence [14]. They also protect the therapeutic against attack by nuclease [15]. By making the nucleic acid stiffer and more rod-like, they are similar to other engineered DNA structures that show enhanced penetration into cells [16]. The minor groove binders do not induce aggregation of nucleic acids as occurs with other delivery reagents such as polyethylenimines where particle size determines distribution in the body and uptake by cells. Some minor groove binders also disrupt membrane structure [17], potentially facilitating escape of therapeutics from endosomes [18].

[ 0007 ] The chemistry of minor groove binders and how to modify them and how to obtain nanomolar binding affinity have been previously reported [14, 19]. Some compounds of this type are already clinically used in humans as anti-parasitic agent in addition to those chemotherapeutics targeting cancer cells [18, 20].

[ 0008 ] An important advance has been the development of methods to synthesize minor groove binding peptides by automated synthesis [21, 22], enabling the use of a wide range of chemistries to modify their properties, including binding specificity and affinity [23]. Design rules have been proposed. For example, an oligomer with n pyrrole units, i.e. n peptide bonds, empirically binds to an (Adenosine or Thymine)_n+1 sequence. While distamycin is specific for 4 bases, a "distamycin trimer" connected head to tail by β-alanine linkers recognizes a 16 base sequence [24], [ 0009] While minor groove binders have been used to increase the strength of association of a triplex forming oligonucleotide (TFO’s) to the major groove of double- stranded DNA, their use in directing the import of nucleic acids into cells has not been reported [25, 26].

[0010] Others have reported the use of peptides to improve the transport of TFO’s into cells but his has not involved minor groove binders [27]. Thus, the efficacy of the use of minor groove binders in targeted therapeutics has yet to be determined.

SUMMARY OF THE INVENTION

[0011] The present invention describes therapeutic compositions for the targeted delivery of nucleic acids to specific cells and tissues, and methods of their use to treat conditions and diseases as described herein. More specifically, the present invention encompasses methods and compositions for the targeted delivery of a therapeutic/drug/pharmaceutical agent, referred to herein as Reagent 1 , to a cell using a synthetic construct referred to herein as a “targetmer”. The invention enables methods known to those skilled in the art that activate, enhance, alter, or inhibit the expression of nucleic acid sequences in the host cell, regardless of whether the expressed sequences derive from Reagent lor from the host nucleic acids. Importantly, the nucleic acids used to construct Reagent 1 are suitable for use in these compositions are unencapsulated.

[ 0012 ] As described herein, the present invention comprises a therapeutic composition comprising at least three components (also referred to herein as “Reagents”. As shown in FIG. 1 , Reagent 1 (“reagent” is also referred to herein as component or agent) is a nucleic acid modified to reduce nuclease susceptibility and wherein the nucleic acid comprises a specific nucleotide sequence designed to elicit or produce a therapeutic effect within a targeted cell or tissue. Reagent 1 has activity to, for example, activate, enhance, alter, or inhibit the expression of nucleic acid sequences in the host cell, thereby eliciting a therapeutic response in the cell or tissue. Reagent 1 is bound/linked/ coupled to one or more copies of a “targetmer”, or to one or more copies of different “targetmers”, wherein a “targetmer” is described in FIG. 1.

[ 0013 ] Modifications to Reagent lto reduce nuclease sensitivity may include the use of modified bases [3]. It may also involve DNAs with covalently closed ends (FIG. 1) [28]. [ 0014 ] As described herein, the targetmer is composed of two parts (also referred to herein as components). The first part is a molecule (Reagent 2) that has sequence-specific binding to the minor-groove of a double-stranded region of a nucleic acid. The second part of the molecule (Reagent 3) is comprised of a ligand that binds specifically to a targeted cell or tissue (e.g., a tumor cell), thereby delivering Reagent 1 to that cell or tissue, resulting in the expression of Reagent 1 in that cell or tissue, and whereby the expression of Reagent 1 results in the desired therapeutic activity of Reagent 1 in in that cell or tissue.

[0015] Together Reagent 2 and Reagent 3 comprise the targetmer.

[ 0016] The combination of Reagent 1 and the targetmer(s) comprise the therapeutic construct/composition.

[0017] The combination of Reagent 1 and the targetmer(s) include design elements for the entry of reagent 1 into the targeted cell or tissue, its release from endosomal compartments, its delivery to the nucleus (if required for efficacy) and the synthesis of RNA it encodes (if required for efficacy).

[ 0018 ] In one embodiment of the targetmer, reagent 2 is covalently coupled to reagent 3, either directly, or via a linker that connects one to the other (Fig. 2a). The different possible methods for forming covalent associations between reagent 2 and reagent 3 are well known to those skilled in the art and are summarized in Farkas and Bystricky [29].

[ 0019] In another embodiment, reagent 2 is coupled to an agent that is has high affinity for another agent coupled to reagent 3, where the association is not covalent, but sufficient to form a targetmer from reagent 2 and reagent 3 (Fig. 2b). The different possible methods for forming non-covalent associations between reagent 2 and reagent 3 are well known to those skilled in the art and are summarized in Table 1 of Schreiber and Smith [30].

[ 0020 ] The binding specificity of reagent 2 to reagent 1 is for a short sequence motif of about 4-16 nucleotides as described herein.

[ 0021 ] The mode of binding of reagent 2 is to the minor groove of double-stranded

DNA.

[0022] The binding kinetics of reagent 2 can be altered by adding nucleotides to reagent 2 so that bind the major groove through base-specific Watson-Crick and Hoogsteen hydrogen bonds. For example, triple helix forming nucleotides bound to minor groove binders are known to change the dissociation rate of a minor groove binder from the nucleic acid [25, 26],

[ 0023 ] Knowing the sequence of reagent 1, it is then possible to determine the exact number of sites at which reagent 2 can bind to reagent 1. This knowledge permits changes to the sequence of reagent 1 to optimize the stoichiometry with which a targetmer binds to reagent 1. Such a metric is desired by regulators responsible for approving the clinical use of therapeutics.

[0024] One embodiment of the invention is when reagent 2 is comprised of molecules known to specifically bind the minor groove, examples of which include distamycin and Hoehsct 33258 (see Fig. 3) [31, 32],

[ 0025 ] Another embodiment of the invention is when reagent 2 is synthesized using solid phase chemistry using pyrrole and imidazole polyamides and their derivatives [14, 21, 22],

[ 0026] Conjugation of reagent 2 to reagent 3 when reagent 3 is a nucleic acid aptamer follows published procedures [33, 34].

[ 0027 ] In another embodiment reagent 3 is a small molecule that has specificity for its ligand and is chemically coupled to reagent 2 through a linker [35], one example being the conjugation of folate or its derivatives to target the folate receptor a. (Fig. 2c).

[ 0028 ] In a particular embodiment, the targetmers are used to deliver Reagent 1 for the purposes of correcting genetic errors that lead to disease. A listing of such errors is given in the online catalog of Mendelian Disease that is entitled “Online Mendelian Inheritance in Man” (known as OMIM, www.omim.org/).

[ 0029] In one embodiment, reagent 3 specifically targets one of the following receptors: VEGFR1, VEGFR2, VEGFR3, HER2/neu, Muc-1, Nucleolin, Optoneurin, Integral, Prostate-Specific Membrane Antigen , AXL, Carcinoembryonic Antigen, folate receptor a , Protein Tyrosine Kinase 7, a checkpoint inhibitor like Cytotoxic T Cell Antigen-4 or Programmed Cell Death Molecule 1 (PD-1), (ASGPR), T Cell Receptors, OX40 and related Tumor Necrosis family members [36], whether with an aptamer [2], carbohydrate, peptide, lipid, steroid protein or small molecule (examples of conjugations to imidazoles are given in Midoux et al. [37]). [ 0030 ] The present invention also encompasses methods for use of targetmers to engineer immune cells ex vivo, such as Chimeric Antigen Receptor (CAR) bearing cells (CAR- cells), then administered to patients to act in vivo.

[ 0031 ] In the case of ex vivo engineered antigen-presenting cells, such as dendritic cells or other myeloid cells, expression of genes delivered using targetmers enhances their antigen-specific function when introduced in vivo, allowing modulation of the immune response against the desired antigen.

[ 0032 ] The present invention also encompasses methods to enhance the effectiveness of tumor-specific vaccines by using targetmers to direct expression of pro-inflammatory proteins IL-23, IL-36γ and OX40L encoded by reagent 1 in antigen presenting cells [38] so as to enhance an immune response against a tumor.

[ 0033 ] The present invention also encompasses methods to enhance the effectiveness of pathogen-specific vaccines by using targetmers to direct expression of pathogen- specific antigen and pro-inflammatory proteins IL-23, IL-36γ and OX40L encoded by reagent 1 in antigen presenting cells [38] so as to enhance an immune response against the pathogen.

[ 0034 ] The present invention encompasses methods to inhibit antigen-specific immune responses, such as those found in autoimmune and allergic diseases by using targetmers to direct expression of inhibitory molecule such as PD- 1 and other checkpoint inhibitors encoded by reagent 1 on cell surfaces bearing the immunostimulatory antigen.

[ 0035 ] Methods described herein include the construction of a therapeutic composition constructed from a nucleic acid encoding therapeutic molecules (reagent 1) that will bind a known number of targetmers.

[ 0036] Methods described herein allow the use of more than one targetmer for the delivery of reagent 1. The targetmers used may differ in reagent 2, reagent 3 or in both, allowing the tuning of the therapeutic delivery for a particular use in a controlled fashion.

[ 0037 ] In one embodiment, reagent 1 derives from a polynucleotide composed of either DNA or RNA that enables expression in the target cell of a desired molecule (Fig. 4). The expression of the fusion protein may be limited to a particular cell type by use of appropriate promoters, enhancers or other regulatory sequences known to one skilled in the art. The therapeutic comprising reagent 1 and the targetmer(s) may be delivered to the target cell by injection, electroporation or other mechanical or electrophysiological mechanisms either locally into a space like the spinal cord or the peritoneum where the therapeutic can contact the targeted tissue. Alternatively, administration may be by systemic administration that involves transport by the blood or lymphatic systems, or after ex-vivo manufacture in the case of a cellular therapy.

[0038 ] The methods of the present invention include an expression vector (reagent 1) coupled to one, or more targetmer(s), wherein the vector comprises a nucleic acid construct that expresses a molecule(s) encoded by reagent 1. As a result of contacting a target cell, reagent 1 is delivered to those cells that binds reagent 2 and results in the transport of reagent 1 to the nucleus.

[0039] In the case where immunosuppression is necessary, the methods include delivery of a defined antigen along with immunosuppressive molecules like PD- 1 and TGFβ decreases or suppresses immune responses associated with allergy and autoimmunity triggered by the specified antigen. Delivery of the defined antigen may be implemented in a number of ways as known to those experienced in the art and does not always depend upon reagent 1.

[0040 ] In a particular embodiment, the subject of the methods of this invention is a mammal, and more particularly, the mammal is a human and can activate immunity using the approaches described.

[0041 ] A particular embodiment of the present invention encompasses methods of treating cancer in an individual, preventing metastasis of the cancer and protecting against reoccurrence of the cancer wherein administering to the individual an effective amount of the therapeutic agent that increases the expression of the encoded molecules targeted to the tumor cells or to cells in the tumor micro-environment. The methods described herein using targeted delivery of reagent 1 by the targetmer(s) can be used to treat many different forms of cancers. For example, the cancer can be ovarian, breast, colon, renal, neural or lung cancer.

[0042 ] Another embodiment of the present invention is a vaccine composition, and method of using that vaccine composition, to vaccinate (i.e., elicit an immune response) a subject against tumors that express a defined antigen so as to provoke an immune response to protect an individual against that tumor type, including applications where the vaccine is delivered locally, to lymph nodes, to other tissues or systemically by injection and targeted to the tumor cells or to cells in the tumor micro-environment.

[0043] Alternatively, the vaccine composition can be used to vaccinate a subject against a pathogen that expresses a defined antigen by targeting the therapeutic to a set of immune cells and so provoke an immune response to protect an individual against any pathogen that bears the antigen.

[0044 ] Also encompassed by the present invention are methods of treating diseases, such as cancer, that further comprise administering the targeted therapeutic composition to a subject concurrently with, or sequentially before or after, or in conjunction with, at least one, or more additional or complementary treatments suitable for the treatment of the specific disease, such as cancer. For example, without limitation, the complementary cancer treatment can be selected from a therapy comprising checkpoint inhibitor; a proteasome inhibitor; immunotherapeutic agent; radiation therapy or chemotherapy. Other suitable additional or complementary cancer therapies are known to those of skill in the art.

[0045] Also encompassed by the present invention is a pharmaceutical composition, or compositions, comprising a therapeutically effective amount of the targeted therapeutic composed of reagents 1 , 2 and 3 as described herein. The composition additionally can include a pharmaceutically acceptable medium, suitable as a carrier for the agent. The compositions can also include other agents that improve delivery of the compositions to specific tumor sites. One embodiment includes the administration of 4- methylumbelliferone to reduce the thickness of the glycocalyx present in tumors.

[0046] The above and other features of the invention including various novel details of construction and combinations of parts, and other advantages, will now be more particularly described with reference to the accompanying drawings and pointed out in the claims. It will be understood that the particular methods and compositions embodying the invention are shown in the drawings and examples by way of illustration and not as a limitation of the invention. The principles and features of this invention may be employed in various and numerous embodiments without departing from the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In the accompanying drawings, reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale; emphasis has instead been placed upon illustrating the principles of the invention. Of the drawings:

[ 0048 ] FIG. 1 : Construction of Nucleic Acid Delivery Therapeutic from Three Reagents, where Reagent 1 is a nucleic acid with closed ends to reduce nuclease susceptibility, Reagent 2 is a molecule that binds to the minor groove of Reagent 1 in a sequence specific manner with micro-molar or sub-micro-molar affinity, Reagent 3 is a ligand for a receptor that is expressed on the targeted cell. Linkers are shown between Reagent 2 and 3 so that the length between each can be varied to improve interaction of Reagent 3 with its receptor.

[ 0049] FIG. 2a-c: Examples where different variations of reagent 2 and reagent 3 are combined to form Nucleic Acid Delivery Therapeutic from reagent 1. Reagent 2 is a molecule that binds to the minor groove of reagent 1 in a sequence specific manner (e.g. Distamycin, Hoechst 33428, netropsin or a polyamide synthesized from a heterocyclic or heteroaryl, aromatic amino acid) and may be covalently linked to reagent 3 or through a high affinity non-covalent interaction (e.g. hybridization by complementary nucleic acid sequences, biotin-avidin interaction), all synthesized by methods well known to those skilled in the art and detailed in the published scientific literature (see for example, WO1996/026950A1, the teachings of which are incorporated herein by reference. Reagent 3 is a ligand for the receptor on the targeted cell (e.g. aptamer, peptide, protein, drug, carbohydrate, vitamin etc.).

[ 0050 ] FIG. 3 : Examples of compounds that bind the minor groove of nucleic acids

[31]·

[ 0051 ] FIG. 4: Example of the various elements in Reagent 1; an enhancer, promoter,

5' UTR (5' untranslated region), intron, INSERT 1 (first element encoded by therapeutic), IRES (Internal ribosome entry site or a 2 A peptide site), INSERT2 (second element encoded by therapeutic, if needed), WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) and a STOP codon. DTS is a DNA targeting sequences that promotes nuclear localization of reagent 1 [39]. [ 0052 ] FIG. 5 : Example of using pyrrole and imidazole polyamides to synthesize a minor-groove binding agent with N- terminal reagent groups to allow coupling to reagent 3 or to a linker capable of a non-co valent attachment to reagent 3. The solid-phase synthesis follows the method of Wurth et al. ([22]. The Fmoc solid phase synthetic scheme for polyamides 1 and 2 starting from commercially available Fmoc-β-alanine-Wang resin: (i) 20% piperidine/NMP; (ii) Fmoc-Py acid, HBTU, DIEA; (iii) 20% piperidine /NMP; (iv) Fmoc-Py acid, HBTU, DIEA; (v) 20% piperidine/NMP ; (vi) Fmoc-Py acid, HBTU, DIEA; (vii) 20% piperidine/NMP; (viii) Fmoc-Py acid (for 1) or Fmoc-Im acid (for 2), HBTU, DIEA; (ix) 20% piperidine/NMP; (x) Fmoc-γ-aminobutyric acid, HBTU, DIEA; (xi) 20% piperidine/NMP; (xii) Fmoc-Py acid, HBTU, DIEA; (xiii) 20% piperidine/NMP; (xiv) Fmoc-Py acid, HBTU, DIEA; (xv) 20% piperidine/NMP; (xvi) Fmoc-Py acid, HBTU, DIEA; (xvii) 20% piperidine/NMP; (xviii) Im-acid, HBTU, DIEA; (xix) N,N- dimethylaminopropylamine, 55 °C. The N-terminal residues are added using commercially available Fmoc reagents.

[ 0053 ] FIG. 6: Example of using pyrrole and imidazole polyamides to synthesize a minor-groove binding agent with Carboxy- terminal reagent groups to allow coupling to reagent 3 or to a linker capable of a non-covalent attachment to reagent 3. The solid-phase synthesis follows the method of Wurth et al. [16] as described in the legend to Fig. 5.

[ 0054 ] FIG. 7 A and B: Chemistry for Fmoc protected Folate for solid phase peptide synthesis using click chemistry (from [40]).

[ 0055 ] FIG. 8: A targetmer for the Folate Receptor a consisting of folate linked to distamycin through a polyethylene glycol linker (molecular weight and is determined by application).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] The invention now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. [ 0057 ] The publications discussed throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.

[ 0058 ] As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. Further, the singular forms and the articles "a", "an" and "the" are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms: includes, comprises, including and/or comprising, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Further, it will be understood that when an element, including component or subsystem, is referred to and/or shown as being connected or coupled to another element, it can be directly connected or coupled to the other element or intervening elements may be present.

[ 0059] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, exemplary methods, and materials are described herein.

[ 0060 ] General texts, which describe molecular biological techniques useful herein, including the use of vectors, promoters and many other relevant topics, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology Volume 152, (Academic Press, Inc., San Diego, Calif.) ("Berger"); Sambrook et al., Molecular Cloning— A Laboratory Manual, 2d ed., Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 ("Sambrook") and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1999) ("Ausubel"). Examples of protocols sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR), the ligase chain reaction (LCR), Q.beta.-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA), e.g., for the production of the homologous nucleic acids of the disclosure are found in Berger, Sambrook, and Ausubel, as well as in Mullis et al. (1987) U.S. Pat. No. 4,683,202; Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press Inc. San Diego, Calif.) ("Innis"); Amheim & Levinson (Oct. 1, 1990) C&EN 36-47; The Journal OfNIH Research (1991) 3: 81-94; Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Nat'l. Acad. Sci. USA 87:

1874; Lomell et al. (1989) J. Clin. Chem 35: 1826; Landegren et al. (1988) Science 241: 1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4:560; Barringer et al. (1990) Gene 89:117; and Sooknanan and Malek (1995) Biotechnology 13: 563-564. Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improved methods for amplifying large nucleic acids by PCR are summarized in Cheng et al. (1994) Nature 369: 684-685 and the references cited therein, in which PCR amplicons of up to 40 kb are generated.

[0061] The term “cell” is used in reference to methods or systems that produce surfaces bearing targeted receptors with or without antigens and are used without respect to species.

[0062] The terms "vector", "vector construct" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA encoding a protein is inserted by restriction enzyme technology. A common type of vector is a "plasmid", which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), or pMAL plasmids (New England Biolabs, Beverly, Mass.), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes.

[ 0063] In one embodiment, the nucleic acid of reagent 1 can be a replication competent vector capable of infecting only replicating tumor cells with particular mutations. In one embodiment, a replication competent vector comprises an internal ribosomal entry site (IRES) 5' to the heterologous polynucleotide encoding, e.g., a cytosine deaminase, miRNA, siRNA, cytokine, receptor, antibody or the like. When the heterologous polynucleotide encodes a non-translated RNA such as siRNA, miRNA or RNAi then no IRES is necessary, but may be included for another translated gene, and any kind of vector can be used. In one embodiment, the polynucleotide is 3' to a sequence that encodes an unrelated protein. In one embodiment, the vector is a capsid free AAV vector capable of nuclear retention as the targeted cell divides [28].

[ 0064 ] The terms "express" and "expression" mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence or RNA sequence. A DNA sequence or RNA sequence is expressed in or by a cell to form an "expression product" such as a protein. The expression product itself, e.g. the resulting protein, may also be said to be "expressed" by the cell. A polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter.

[ 0065] The terms “gene editing” or “gene editing techniques” as described herein can include RNA-mediated interference (referred to herein as RNAi, or interfering RNA molecules), or Short Hairpin RNA (shRNA) or CRISPR-Cas9 and TALEN. See e.g., Agrawal. N. et al., Microbiol Mol Biol Rev. 2003 Dec; 67(4): 657—685; Moore, C.B., et al. Methods Mol Biol. 2010; 629: 141—158; Doudna, J.A. and Charpentier, E. Science vo. 346, 28 Nov. 2014; Sander, J.D. and Joung, K. Nature Biotech 32, 347-355 (2014); U.S. Pat 8,697,359; Nemudryo, A.A. ACTA Naturae vol. 6, No. 3(22)2014. Anti-sense RNA can also be used. (Gleave, M. and Monia, B., Nature Reviews Cancer 5, 468-479 (June 2005)). The term “gene therapy” generally means a method of therapy wherein a desired gene/genetic sequence is inserted into a cell or tissue (along with other sequences necessary for the expression of the specific gene). See, for example, genetherapynet.com for description of gene therapy techniques.

[0066] The term "subject" as used herein can include a human subject for medical purposes, such as for the treatment of an existing disease, disorder, condition or the prophylactic treatment for preventing the onset of a disease, disorder, or condition or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, gibbons, chimpanzees, orangutans, macaques and the like; bovines, e.g., cattle, oxen, and the like; o vines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, guinea pigs, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a "subject" can include a patient afflicted with or suspected of being afflicted with a disease, disorder, or condition. Thus, the terms "subject" and "patient" are used interchangeably herein. Subjects also include animal disease models (e.g., rats or mice used in experiments, and the like).

[ 0067 ] The term "cancer" or “tumor” includes, but is not limited to, solid tumors and blood borne tumors. These terms include diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. These terms further encompass primary and metastatic cancers.

[ 0068 ] The term “minor groove” refers to the narrower of the two grooves found in structures of double-stranded nucleic acids. Functionally, the minor groove presents a different binding surface for molecules than does the larger “major groove”. Some examples of molecules that specifically recognize the minor groove are given in Lauria et al. [31].

[ 0069] The term “minor groove binder” refers to a compound that binds to the minor groove of double-stranded nucleic acids (Fig. 3) [23, 31]. [0070] The term “targetmer” refers to the combination of a minor-groove binder specific for a double-stranded nucleic-acid (Reagent 2) and a receptor binding ligand (Reagent 3) regardless of how or where they associate.

[ 0071 ] The term “antigen” is defined as any molecule that a T-Cell or B-Cell receptor has specificity for, or any molecule targeted by Natural Killer Cells or other Innate Cells that specifically targets their effector function such as cytotoxic killing of cells, release of growth factors, lymphokines or cytokines. (Microbiology and Immunology On-line, Edited by Richard Hunt, PhD; www.microbiologvbook.org/maver/antigens2000 ).

[ 0072 ] The term “CAR” refers to any chimeric antigen receptor introduced into immune cells for therapeutic purposes [41].

[ 0073 ] The term “genetic disease” includes any of those listed in the online catalogue of Mendelian Disease that is entitled “Online Mendelian Inheritance in Man” (known as OMIM, www.omim.org/).

1. The methods and compositions of the present invention are suitable to treat a disease due to a nucleotide variant in the genome regardless of whether the variant is inherited or arises from a somatic DNA mutation. More specifgically, methods and compositions of the present invention enable treatment of many different types of genetic disease, either to cure the disease or to completely, or partially, ameliorate disease symptoms and its effects.

[ 0074 ] The methods and compositions of the present invention may be used to treat any type cancerous tumor or cancer cells. Such tumors/cancers may be located anywhere in the body, including without limitation in a tissue selected from brain, colon, urogenital, lung, renal, prostate, pancreas, liver, esophagus, stomach, hematopoietic, breast, thymus, testis, ovarian, skin, bone marrow and/or uterine tissue. Cancers that may treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[ 0075] A “therapeutically effective” amount as used herein refers to an amount sufficient to have the desired biological effect (for example, an amount sufficient to express a molecule or molecules with the desired effect on the underlying disease state (for example, an amount sufficient to inhibit tumor growth in a subject, produce an immune response to an antigen or to inhibit autoimmune disease) in at least a subpopulation of cells in a subject at a reasonable benefit/risk ratio applicable to any medical treatment. Determination of therapeutically effective amounts of the agents used in this invention, can be readily made by one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. The amounts/ dosages may be varied depending upon the requirements of the subject in the judgment of the treating clinician; the severity of the condition being treated and the particular composition being employed. In determining the therapeutically effective amount, a number of factors are considered by the treating clinician, including, but not limited to: the specific disease state; pharmacodynamic characteristics of the particular agent and its mode and route of administration; the desired time course of treatment; the species being treated; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual patient; the particular agent administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the kind of concurrent treatment (i.e., the interaction of the agent with other co-administered agents); and other relevant circumstances.

[ 0076] For example, as described herein, the sequence of reagent 1 may be changed to alter the number of binding sited for reagent 3. The composition of reagent 2 may be changed to target different sequence motifs in reagent 1. The composition of reagent 3 may be changed to target reagent 1 to different tissues or cells. Different chemistries known to those skilled in the art may be used to link reagent 2 to either reagent 3, the preferred embodiment, or to reagent 1 (see for example, WO1996/026950A1, the teachings of which are incorporated herein by reference).

[ 0077 ] In certain embodiments, the agents described for use in this invention can be combined with other pharmacologically active compounds ("additional active agents") or peptide antigens (“antigens”) known in the art according to the methods and compositions provided herein. Additional active agents can be large molecules (e.g., proteins, lipids, carbohydrates) or small molecules (e.g., synthetic inorganic, organometallic, or organic molecules). In one embodiment, additional active agents independently or synergistically help to treat cancer.

[ 0078 ] For example, certain additional active agents are anti-cancer chemotherapeutic agents. The term chemotherapeutic agent includes, without limitation, platinum-based agents, such as carboplatin and cisplatin; nitrogen mustard alkylating agents; nitrosourea alkylating agents, such as carmustine (BCNU) and other alkylating agents; antimetabolites, such as methotrexate; purine analog antimetabolites; pyrimidine analog antimetabolites, such as fluorouracil (5-FU) and gemcitabine; hormonal antineoplastics, such as goserelin, leuprolide, and tamoxifen; natural antineoplastics, such as taxanes (e.g., docetaxel and paclitaxel), aldesleukin, interleukin-2, etoposide (VP- 16), interferon alfa, and tretinoin (ATRA); antibiotic natural antineoplastics, such as bleomycin, dactinomycin, daunorubicin, doxorubicin, and mitomycin; and vinca alkaloid natural antineoplastics, such as vinblastine and vincristine or agents targeted at specific mutations within tumor cells.

[ 0079] Further, the following drugs may also be used in combination with an antineoplastic agent, even if not considered antineoplastic agents themselves: dactinomycin; daunorubicin HC1; docetaxel; doxorubicin HC1; epoetin alfa; etoposide (VP- 16); ganciclovir sodium; gentamicin sulfate; interferon alfa; leuprolide acetate; meperidine HC1; methadone HC1; ranitidine HC1; vinblastin sulfate; and zidovudine (AZT). For example, fluorouracil has recently been formulated in conjunction with epinephrine and bovine collagen to form a particularly effective combination.

[0080] Still further, the following listing of amino acids, peptides, polypeptides, proteins, polysaccharides, and other large molecules may also be expressed from reagent 1 or be administered as protein therapeutics along with the therapeutic comprised of reagent 1, 2 and 3: checkpoint inhibitors that target for example, PD-1 and CTLA-4, interleukins 1 through 37, including mutants and analogues; interferons or cytokines, such as interferons .alpha., .beta., and .gamma.; hormones, such as luteinizing hormone releasing hormone (LHRH) and analogues and, gonadotropin releasing hormone (GnRH); growth factors, such as transforming growth factor-. beta. (TGF-.beta.), fibroblast growth factor (FGF), nerve growth factor (NGF), growth hormone releasing factor (GHRF), epidermal growth factor (EGF), fibroblast growth factor homologous factor (FGFHF), hepatocyte growth factor (HGF), and insulin growth factor (IGF); tumor necrosis factor-.alpha. & .beta. (TNF- . alpha. & .beta.); invasion inhibiting factor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7); somatostatin; thymosin-. alpha.- 1 ; . gamma. -globulin; superoxide dismutase (SOD); complement factors; anti-angiogenesis factors; antigenic materials; and pro-drugs.

[0081] Chemotherapeutic agents for use with the compositions and methods of treatment described herein include, but are not limited to alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine ; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esombicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as firolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirambicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[ 0082 ] The compositions and methods of the invention can comprise or include the use of other biologically active substances, including therapeutic drugs or pro-drugs, for example, other chemotherapeutic agents or antigens useful for cancer vaccine applications. Various forms of the chemotherapeutic agents and/or additional active agents may be used. These include, without limitation, such forms as uncharged molecules, molecular complexes, salts, ethers, esters, amides, and the like, which are biologically active.

[ 0083 ] The agents and substances described herein can be delivered to the subject in a pharmaceutically suitable, or acceptable or biologically compatible carrier. The terms “pharmaceutically suitable/ acceptable” or “biologically compatible” mean suitable for pharmaceutical use (for example, sufficient safety margin and if appropriate, sufficient efficacy for the stated purpose), particularly as used in the compositions and methods of this invention.

[ 0084 ] The compositions described herein may be delivered by any suitable route of administration for treating the cancer, including orally, nasally, transmucosally, ocularly, rectally, intravaginally, parenterally, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articular, intra- stemal, intra- synovial, intra-hepatic, through an inhalation spray, or other modes of delivery known in the art.

[ 0085 ] The nucleic acid sequence for VEGFR1 (FLT1) can be found at “fins related receptor tyrosine kinase 1 [ Homo sapiens (human) ]” Gene ID: 2321, www.ncbi.nlm.nih.gov/gene, updated on 7-Dec-2019 , the nucleic acid sequence for VEGFR2 (KDR) can be found at “kinase insert domain receptor [ Homo sapiens (human)]” Gene ID: 3791, www.ncbi.nlm.nih.gov/gene, updated on 7-Dec-2019, the nucleic acid sequence for VEGFR3 (FLT4) can be found at “fins related receptor tyrosine kinase 4 [ Homo sapiens (human) ]” Gene ID: 2324, www.ncbi.nlm.nih.gov/gene, updated on 7- Dec-2019, the nucleic acid sequence for HER2/neu (ERBB2) can be found at “erb-b2 receptor tyrosine kinase 2 [ Homo sapiens (human) ]” Gene ID: 2062, www.ncbi.nlm.nih.gov/gene, updated on 7-Dec-2019 , the nucleic acid sequence for folate receptor a (FOLR1) can be found at “folate receptor alpha [ Homo sapiens (human) ]” Gene ID: 2348, www.ncbi.nlm.nih.gov/gene, updated on 7-Dec-2019, the nucleic acid sequence for Muc-1 (MUC1) can be found at “mucin 1, cell surface associated [ Homo sapiens (human) ]” Gene ID: 4582, www.ncbi.nlm.nih.gov/gene, updated on 7 -Dec-2019 , the nucleic acid sequence for Nucleolin (NCL) can be found at “nucleolin [ Homo sapiens (human) ]” Gene ID: 4691, www.ncbi.nlm.nih.gov/gene, updated on 7 -Dec-2019, the nucleic acid sequence for Optoneurin (OTN) can be found at “Optoneurin [ Homo sapiens (human) ]” Gene ID: 10133, www.ncbi.nlm.nih.gov/gene, updated on 7-Dec-2019, the nucleic acid sequence for Prostate-Specific Membrane Antigen (FOLH1) can be found at “folate hydrolase 1 [ Homo sapiens (human) ]” Gene ID: 2346, www.ncbi.nlm.nih.gov/gene, updated on 7-Dec-2019, the nucleic acid sequence for Prostate- Specific Membrane Antigen (AXL) can be found at “AXL receptor tyrosine kinase [ Homo sapiens (human) ]” Gene ID: 558, www.ncbi.nlm.nih.gov/gene, updated on 7-Dec-2019, the nucleic acid sequence for Carcinoembryonic Antigen (| can be found at “CEA cell adhesion molecule 5 [ Homo sapiens (human) ]” Gene ID: 1048, www.ncbi.nlm.nih.gov/gene, updated on 7-Dec-2019, the nucleic acid sequence for Protein Tyrosine Kinase 7 (PTK7) can be found at “protein tyrosine kinase 7 (inactive) [ Homo sapiens (human) ]” Gene ID: 5754, www.ncbi.nlm.nih.gov/gene, updated on 7 -Dec-2019, the nucleic acid sequence for the checkpoint inhibitor like Cytotoxic T Cell Antigen-4 (CTLA4) can be found at “cytotoxic T-lymphocyte associated protein 4 [ Homo sapiens (human) ]” Gene ID: 1493, www.ncbi.nlm.nih.gov/gene, updated on 7 -Dec-2019, the nucleic acid sequence for the checkpoint inhibitor Programmed Cell Death Molecule 1 (PDCD1) can be found at “programmed cell death 1 [ Homo sapiens (human) ]” Gene ID: 5133, www.ncbi.nlm.nih.gov/gene, updated on 7-Dec-2019, the nucleic acid sequence for the checkpoint inhibitor Programmed Cell Death Molecule 1 Ligand 1 (CD274) can be found at “CD274 molecule [ Homo sapiens (human) ]” Gene ID: 29126, www.ncbi.nlm.nih.gov/gene, updated on 7-Dec-2019, the nucleic acid sequence for the checkpoint inhibitor Programmed Cell Death Molecule 1 Ligand 2 (PDCD1LG2) can be found at “programmed cell death 1 ligand 2 [ Homo sapiens (human) ]” Gene ID: 80380, www.ncbi.nlm.nih.gov/gene, updated on 7-Dec-2019, the nucleic acid sequence for asialoglycoprotein receptor (ASGPR) can be found at asialoglycoprotein receptor 1

[ Homo sapiens (human) ]” Gene ID: 432, www.ncbi.nlm.nih.gov/gene, updated on 7- Dec-2019 , the nucleic acid sequence for OX40 (TNFRSF4)can be found at “TNF receptor superfamily member 4 [ Homo sapiens (human) ]” Gene ID: 7293, www.ncbi.nlm.nih.gov/gene, updated on 7-Dec-2019, the T Cell Receptor Alpha chain (TRA) can be found at “T cell receptor alpha locus [ Homo sapiens (human) ]” Gene

ID: I, www.ncbi.nlm.nih.gov/gene, updated on 7-Dec-2019, the genes for the T Cell Beta Chain (TRB) can be found at “T cell receptor beta locus [ Homo sapiens (human) ]”

Gene ID: I, www.ncbi.nlm.nih.gov/gene, updated on 7-Dec-2019, the genes for the T Cell delta chain (TRD) can be found at “T cell receptor delta locus [ Homo sapiens (human) ]” Gene ID: I, www.ncbi.nlm.nih.gov/gene, updated on 7 -Dec-2019, and the genes for the T Cell Gamma chain (TRG) can be found at “T cell receptor gamma locus [ Homo sapiens (human) ]” Gene ID: 6965, www.ncbi.nlm.nih.gov/gene, updated on

7-Dec-2019.

[ 0086] For example, a gene editing technique to alter genomic DNA sequences within tumors can be used so that the protein product is targeted to the cell surface membrane as described in this invention (see e.g., US Patent 8,697, 359 for a description of CRISPR techniques). Delivery of CRISPR/CAS9 with a sgRNAs to a tumor cell and other sequences necessary to effect the desired change following cleavage of the targeted DNA can be provided by use of Reagent 1. A number of vectors have been used in humans and these can be used to express the genetic material in different cell types. Such methods are known to those of skill in the art. Means to target expression of the receptors that targetmers have affinity for are also known to those of skill. For example, genetically engineered vectors exist that direct the expression of receptors that facilitate targetmer delivery into a particular cell type. An example is given in Figure 3. This construct also includes a reporter gene that allows efficiency of transduction of the virus into the tumor to be quantitated.

[ 0087 ] The above approaches can be combined with other cancer therapies including immune-modulators such as checkpoint inhibitor ligands for PD-1 CTLA-4, ICOS, OX40;; lymphokines, cytokines and their receptors and strategies designed to increase major and minor histocompatibility antigens. Additionally, the methods of the present invention can be combined with other standard cancer therapies such as radiotherapy and chemotherapy. [ 0088 ] The above approaches can be used to deliver nucleic acids for the repair genetic defects underlying Mendelian disease and others that are the product of a DNA mutation or a change in genomic sequence that occurs only in somatic cells.

[ 0089] Examples

General Description of the Therapeutic:

The targeted therapeutic composition described herein comprises three parts:

1. Reagent 1 that carries nucleic acid sequences necessary to produce the therapeutic effect;

2. Reagent 2 that is a molecule that binds to the minor groove of a double-stranded nucleic acid; and

3. Reagent 3 that is a ligand specific for a receptor(s) on the targeted cell.

[ 0090 ] Additionally, the composition comprises a means of attaching Reagent 2 to Reagent 3 — either through a covalent chemical bond or by a non-covalent association. The attachment may or may -not involve a linker.

[0091] Example 1 : Targetmer Synthesis

[ 0092 ] The targetmer is synthesized using solid state peptide synthesis to create distamycin and related minor groove binders derived from pyrrole— imidazole (Py— Im) polyamides by solid phase synthesis, most commonly using Fmoc chemistry to create compounds of different length and composition [23]. A number of variations to this approach are possible that include incorporation of heteroaryl ring compounds that are comprised of 5 to 14 atoms, including thienyl, furyl, pyrrolyl, indolyl, pyrimidinyl, isoxazolyl, purinyl, imidazolyl, pyridyl, pyrazolyl, quinolyl, pyrazinyl and their derivatives in order to optimize the properties of the minor-groove binding compound for nucleic acid delivery. For instance, the incorporation of histidine may make certain properties of the compound pH dependent to enable release of reagent 1 from endosomes [42] while the incorporation of tryptophan may increase its hydrophobicity to promote incorporation into membranes [43]. [ 0093 ] Reactive groups for covalent coupling of Reagent 2 to Reagent 3 are introduced during chemical synthesis of reagent 2. In scheme 1 they are introduced to the N-terminus at the end of synthesis using standard FMOC chemistry that enables the introduction of azido-homoalanine, propargyl glycine, formyl glycine, thiol [44] as well as a primary amine (Fig. 4). In scheme 2, they can be introduced by attachment of a modified amino acid bearing 3 -amino-propylazide or propargyl functional groups as first described by Barany and modified by Ten Brink et al. [45, 46] (Fig. 6). Reagent 3 bearing the appropriate reactive group can then be coupled to the modified peptide. Most commonly the coupling will be through an amino group using a NHS ester or by reductive amidation of an aldehyde group. Alternatively coupling via click chemistry involving either an azide or alkyne group will be the preferred embodiment (Fig. 7 A and B). An example of a folate receptor a receptor targetmer with distamycin A linked through a polyethylene glycol linker to folate is shown in FIG. 8.

[ 0094 ] Example 2: Reagent 3 Constructs

[ 0095 ] Reagent 3 can be constructed from published aptamer sequences that are specific for the listed ligand receptors in vivo. Nucleotide residues are shown as single letters with DNA sequences containing “T” for thymidine and RNA sequences containing “U” for uridine, “A“= adenine, “G” = Guanine and “C” = cytidine. Kd stands for the dissociation constant. The sequence, the target, the reported dissociation constant and the reference are given.

5’-TGTGGGGGTGGACGGGCCGGGTAGA-3' (VEGF, Kd=1.2 nM, [47]) (SEQ ID NO:l)

5'-GGGTTATATTACTCGGCCGGTGTAA-3' (Mucin-1, Kd= 25 nM, [48]) (SEQ ID NO:2)

5'-CTCGTCCCCCAGGCATAGATACTCCG CCCCGTCACGGATCCTCTAGAGCA -3' (Annexin A2, Kd not given, [49]) (SEQ ID NO:3)

5'-CCCCCCGGGGCGGGCCGGGGGCGGGGTCCCGGCGGGGCGGAGCCATG -3' (Nucleolin, Kd= 79 nM, [50]) (SEQ ID NO:4)

S'-GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCU -3' (Prostate Surface Membrane Antigen (PSMA), Kd=10 nM, [51]) (SEQ ID NO:5) 5'-CGGCCACAGAAUGAAAAACCUCAUCGAUGUUGCAUAGUUG-3' (Optoneurin, Kd = 18 nM , [52]) (SEQ ID NO:6)

[ 0096] A listing of other aptamers is provided in Table 1 of Morita et al [53] . Generation of additional aptamers with novel sequences and different affinities for cell surface receptors is routine for those skilled in the art and based on the SELEX method

[54],

[ 0097 ] Example 3: Suitable Peptide Sequences

[ 0098 ] Peptide Sequences published in the Scientific Literature that are specific for cell surface receptors in vivo as suitable for use as Reagent 3. A listing of sequences and targets are given in Table 1 of Kang et al. [55] and in Morita et al [53], along with references.

[0099] Example 4: Suitable Small Molecules

[00100] Small molecules from the published literature that are specific for cell surface receptors in vivo are suitable for use as Reagent 3. A list of those in clinical trials is provided by Srinivasarao et al. [56], along with descriptions of the chemistries available for linking them to other molecules. Folate is commonly used [40].

[00101] Example 5 : Reagent 3 : Antibody

[00102] Reagent 3 may be an antigen-specific antibody, a lectin specific for abnormal glycoproteins on a cancer cell (for example N -gly colylneuraminic acid by the B submit of the subtilase cytotoxin) antibodies specific for viral proteins (for example, antibodies derived from individuals immune to a particular virus) a nucleic acid, modified or not, that can bind sequence specifically to another nucleic acid.

[00103] Reagent 1 can be combined with different targetmers. For example, the targetmer specific for a cell surface receptor may be used in conjunction with other targetmers composed of a nucleic sequence or drug that inhibits Toll-like Receptor (TLR) activation of immune responses. For example, one of the targetmer may incorporate telomere sequences to inhibit TLR9, or GpC sequences to inhibit TLR9 [57], or small molecules to inhibit TLR4 [58, 59],

[ 00104 ] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

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Claims

CLAIMS What is claimed is:

1. A targetmer composition comprised of two components: a), a minor-groove binding polyamide sequence specific for a double stranded nucleic acid; and b). a receptor binding ligand.

2. The targetmer of claim 1, wherein the minor-groove binding polyamide sequence and the receptor binding ligand are linked.

3. The targetmer of claim 2, wherein the minor-groove binding polyamide sequence and the receptor binding ligand are covalently linked.

4. The targetmer of claim 2, wherein the minor-groove binding polyamide sequence and the receptor binding ligand are linked by non-covalent association.

5. A therapeutic composition comprising a therapeutic nucleic acid molecule bound to one, or more targetmers of claims 1 -4.

6. The method of claim 5 where the therapeutic nucleic acid molecule is modified to resist nucleases or other enzymes that degrade it, either chemically or through closed ends.

7. A pharmaceutical composition comprising the therapeutic composition of claim 5.

8. A method of enhancing the delivery of a therapeutic nucleic acid (reagent 1) to a subject by using targetmers, the method comprising contacting a cell with an targetmer consisting of a part that binds the minor groove of a double-stranded nucleic acid (reagent 2) and a part that targets the complex to a cell surface (reagent 3) wherein the targetmer increases the delivery and expression of sequences encoded by the nucleic acid in the cell contacted by the targetmer.

9. The method of claim 8 wherein the reagent 1 is a DNA vector that directs the expression of one or more nucleic acids suitable to treat a disease due to a nucleotide variant in the genome regardless of whether the variant is inherited or arises from a somatic DNA mutation.

10. The method of claim 8 is a nucleic acid containing modified bases that inhibits the expression of an RNA in a cell or recodes its sequence.

11. The method of claim 8 wherein reagent 1 comprises a gene-editing agent that alters the genomic sequence.

12. The method of either claim 10 or 11, wherein the gene-editing agent comprises a

CRISPR-Cas system construct that alters a protein or its level of expression within the cell, on the cell surface or promotes its release into the extra-cellular milieu.

13. The method of either claim 10 or 11, wherein the gene-editing agent comprises a

TALEN construct that alters a protein or its level of expression within the cell, on the cell surface or promotes its release into the extra-cellular milieu.

14. The method of either claim 10 or 11, wherein the gene-editing agent comprises a meganuclease construct that alters a protein or its level of expression within the cell, on the cell surface or promotes its release into the extra-cellular milieu.

15. The method of either claim 10 or 11, wherein the gene-editing agent comprises a recombinase construct that alters a protein or its level of expression within the cell, on the cell surface or promotes its release into the extra-cellular milieu.

16. The method of either claim 10 or 11, wherein the gene-editing agent comprises a baseediting construct that alters a protein or its level of expression within the cell, on the cell surface or promotes its release into the extra-cellular milieu.

17. The method of claim 8, wherein reagent 2 is derived by chemical synthesis using imidazole, pyrrole, thienyl, furyl, pyrrolyl, indolyl, pyrimidinyl, isoxazolyl, purinyl, imidazolyl, pyridyl, pyrazolyl, quinolyl or pyrazinyl derivatives for its stepwise production.

18. The method of claim 8, wherein reagent 2 is derived by chemical modification of a drug known to bind the minor groove of a double-stranded nucleic acid to allow association with reagent 3.

19. The method of claim 8, wherein reagent 3 is a ligand that binds specifically to a cell surface, regardless of whether its chemical composition consists of a nucleic acid, a peptide, an antibody, a carbohydrate, lipid, steroid, a small molecule or a drug.

20. The method of claim 19 where reagent 3 is chemically modified to resist nucleases, proteases or other enzymes that degrade reagent 3.

21. The method of claim 8, wherein reactive groups are introduced to reagent 2 that enable covalent coupling to reagent 3.

22. The method of claim 8, wherein groups are introduced to reagent 2 that enable non- covalent coupling to reagent 3.

23. The method of claim 8, wherein the subject is a mammal.

24. The method of claim 8, wherein the mammal is a human.

25. The method of claim 8, wherein the therapeutic comprises an expression vector that targets a tumor cell, wherein the vector comprises a nucleic acid construct that produces inhibition of tumor growth.

26. The method of claim 25, wherein the cancer treatment is administration of a treatment selected from the group consisting of: a checkpoint inhibitor; a proteasome inhibitor; immunotherapy; radiation therapy; chemotherapy.

27. The method of claim 25, wherein the cancer treatment is a vaccine directing the expression or activity of a tumor specific antigen or a biologically active variant thereof including peptides derived from the tumor cell or the tumor cell microenvironment to provoke a specific immune response against the tumor.

28. The method of claim 8, wherein the therapeutic comprises a targetmer specific for a cell involved in an inflammatory response that is producing tissue damage, wherein reagent 1 comprises a nucleic acid construct encoding RNAs or proteins that inhibits the immune response.

29. The method of claim 28, wherein the treatment is a vaccine inhibiting the immunological response to a specific antigen.

30. The method of claim 8, wherein the therapeutic comprises a targetmer specific for a virally infected cell, wherein reagent 1 comprises a nucleic acid construct encoding RNAs or proteins that inhibit or destroy the virus.

31. The method of claim 8, wherein the therapeutic comprises a targetmer that targets a virally infected cell, wherein reagent 1 comprises a nucleic acid construct encoding RNAs or proteins that stimulate an anti-viral immune response.

32. The method of claim 8, wherein the agent is used to produce a CAR-bearing cell in vitro to be used in vivo to modulate an immune response.