Patent Application
20240285804
Described herein are compositions and methods for tissue-targeted expression of therapeutic genes, using AAV expression vectors.
For tissue-targeted gene therapy, e.g., in the CNS, it is desirable to use a vector that is targeted to the specific tissue and has reduced expression in non-target tissues.
Provided herein are AAV vectors comprising an AAV capsid, wherein the AAV capsid comprises a peptide insert of up to 21 amino acids, and wherein the peptide insert comprises a targeting peptide as described herein, e.g., 5-7 amino acids of a peptide shown in Table A, e.g., TVSALFK (SEQ ID NO: 36); a microRNA targeting sequence, preferably at the 3′UTR; and optionally a transgene, e.g., a therapeutic transgene. In some embodiments, the peptide insert comprises TVSALFK (SEQ ID NO:36)(also referred to herein as targeting peptide CPP.21). In some embodiments, the peptide insert comprises TVSALK (SEQ ID NO:32) (also referred to herein as targeting peptide CPP.16). In some embodiments, the peptide insert consists of TVSALFK (SEQ ID NO: 36). In some embodiments, the peptide insert consists of TVSALK (SEQ ID NO: 32). In some embodiments, the AAV vector comprises a transgene sequence. In some embodiments, the transgene sequence encodes a therapeutic agent (e.g., a therapeutic transgene).
In some embodiments, the AAV vector comprises a non-coding RNA, e.g., an shRNA, siRNA or miRNA.
In some embodiments, delivery of the transgene sequence or the non-coding RNA to an organ or tissue is enhanced relative to an AAV vector comprising an AAV capsid without the peptide insert and the transgene sequence or the non-coding RNA. In some embodiments, the organ or tissue comprises permeability barriers. In some embodiments, the organ or tissue comprises epithelium comprising tight junctions. In some embodiments, the organ or tissue is the brain or central nervous system.
Also provided are compositions comprising the AAV vector of claim 2, and a pharmaceutically acceptable carrier.
Further, provided herein are adeno-associated virus (AAV) vectors, preferably comprising a target tissue-tropic capsid (e.g., comprising a targeting peptide, e.g., comprising 5-7 amino acids of a peptide shown in Table A, e.g., TVSALFK (SEQ ID NO:36), optionally AAV.CPP.16, AAV.CPP.21 or AAV9, and further comprising an expression cassette comprising, preferably from 5′ to 3′: a promoter that drives expression in cells of the target tissue, an optional secretory signal peptide sequence, a coding sequence for a protein of interest, an optional Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), a polyA signal sequence, and at least one microRNA targeting sequence selected from a microRNA 122 targeting sequence (miR-122T), e.g., targeting the 5p strand, e.g., comprising CAAACACCATTGTCACACTCCA (SEQ ID NO: 21); a microRNA 124 targeting sequence (miR-124T), e.g., targeting the 5p strand, e.g., comprising ATCAAGGTCCGCTGTGAACACG (SEQ ID NO: 20); a microRNA 200c targeting sequence (miR-200cT), e.g., targeting the 5p strand, e.g., CCAAACACTGCTGGGTAAGACG (SEQ ID NO: 22); and/or microRNA 1 targeting sequence (miR-1T), e.g., targeting the 5p strand, e.g., ATGGGCATATAAAGAAGTATGT (SEQ ID NO: 24), at the 3′ UTR. In some embodiments, the peptide insert comprises TVSALFK (SEQ ID NO:36). In some embodiments, the peptide insert comprises TVSALK (SEQ ID NO: 32). In some embodiments, the peptide insert consists of TVSALFK (SEQ ID NO: 36). In some embodiments, the peptide insert consists of TVSALK (SEQ ID NO: 32).
In some embodiments, the capsid is CNS tropic, e.g., neuronal or glial-tropic.
In some embodiments, the promoter that drives expression in the CNS drives expression in neuronal cells or glial cells. In some embodiments, the promoter that drives expression in glial cells is a GFAP promoter, gfaABC1D promoter, gfa2 promoter, ALDH1L1 promoter, SLC1A3 promoter, Gjb6 promoter, Mbp promoter, MAG promoter, CBh promoter, F4/80 promoter, CD68 promoter, or CD11B promoter.
In some embodiments, the promoter that drives expression in neuronal cells is a neuronal-specific enolase (NSE) promoter, Synapsin promoter, calcium/calmodulin-dependent protein kinase II promoter, tubulin alpha 1 promoter, platelet-derived growth factor beta chain promoter, parvalbumin promoter, GAD67 promoter or CCK promoter.
In some embodiments, the promoter is a ubiquitous promoter, optionally major immediate early human cytomegalovirus promoter (MIEhCMV), Chicken R-Actin Promoter (CBA); Human Cytomegalovirus Immediate/Early Gene Promoter and Enhancer (CMV); Chicken β-Actin/Cytomegalovirus Hybrid Promoter (CAG); Rous Sarcoma Virus Long Terminal Repeat Promoter (RSV); SV40 promoter; EF1alpha promoter.
In some embodiments, the AAV vectors further comprise an enhancer, optionally CMV-Enhancer, mDlx enhancer, AQP4 enhancer.
In some embodiments, the optional secretory signal peptide sequence is a Human IL-2 signal peptide (optionally MYRMQLLSCIALSLALVTNS, SEQ ID NO: 16), human albumin signal peptide, human alpha 1-antitrypsin signal peptide, or human factor VIII signal peptide.
In some embodiments, the polyA signal sequence is from human growth hormone (hGH), SV40, bovine growth hormone (bGH), or beta-globin, e.g., rabbit beta-globin (rbGlob).
In some embodiments, the AAV vectors include a plurality of, e.g., 2-10 or 3-5, microRNA targeting sequences, optionally separated by spacer sequence (e.g., of 1-50 nucleotides).
In some embodiments, the protein of interest is a therapeutic protein. In some embodiments, the therapeutic protein of interest is an antibody such as immune checkpoint inhibitors.
In some embodiments, the therapeutic protein of interest is a toxin, a suicide protein, or an antibody. In some embodiments, the toxin is diphtheria toxin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), or TNF-α. In some embodiments, the suicide protein is herpes simplex virus thymidine kinase (HSVTK), bacterial or fungal cytosine deaminase (CD), carboxypeptidase G2 (CPG2), nitroreductase (NTR), Cytochrome P450 (CYP), purine nucleoside phosphorylase (PNP), horseradish peroxidase (HRP), or carboxylesterase (CE).
Also provided herein are methods of directing expression of a protein of interest in a cell in a target tissue, preferably without substantial expression of the protein of interest in non-target cells, the method comprising introducing an AAV vector as described herein into the cell.
Additionally provided are methods of inducing cell death in a cell, e.g., in a glial cell or a neuronal cell, the method comprising introducing an AAV vector as described herein into the cell, preferably wherein the therapeutic protein of interest is a toxin, a suicide protein, or an antibody. In some embodiments, the therapeutic protein of interest is a suicide protein, and the method further comprises contact the cell with a nontoxic prodrug that is a substrate for the suicide protein, wherein action of the suicide protein on the nontoxic prodrug results in production of a toxic metabolite that induces cell death. In some embodiments, the suicide protein is herpes simplex virus thymidine kinase (HSVTK) and the nontoxic prodrug is ganciclovir (GCV); the suicide protein is cytosine deaminase (CD) and the nontoxic prodrug is 5-flourouracil (5-FU); the suicide protein is carboxypeptidase G2 (CPG2) and the nontoxic prodrug is nitrogen mustard (NM) or a derivate thereof such as ZD2767P or CMDA (4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamicacid); the suicide protein is nitroreductase (NTR) and the nontoxic prodrug is CB1954 or an analog thereof; the suicide protein is Cytochrome P450 (CYP) and the nontoxic prodrug is an oxazaphosphorine drug such as cyclophosphamide (CPA) and ifosfomide (IFO); the suicide protein is purine nucleoside phosphorylase (PNP) and the nontoxic prodrug is 6-Methylpurine Deoxyriboside or an analog thereof, e.g., fludarabine phosphate (F-araAMP) or 2-fluoro-2-deoxyadenosine (F-dAdo); the suicide gene is horseradish peroxidase (HRP) and the nontoxic prodrug is indole-3-acetic acid (HRP/IAA); or the suicide protein is carboxylesterase (CE) and the nontoxic prodrug is irinotecan.
In some embodiments, the glial cell is a cancer cell in a subject. In some embodiments, the cancer is glioblastoma.
In some embodiments, the AAV vector is a vector that targets the CNS; wherein the capsid is AAV.CPP.16 or AAV.CPP.21; the promoter is a GFAP promoter or a Syn promoter; and the microRNA targeting sequences target one, two or all three of microRNA 122, microRNA 200c and microRNA 1. In some embodiments, the protein of interest is MeCP2, CNTF, or NGF.
In some embodiments, the AAV vector is a vector that targets the Liver; wherein the capsid is AAV.CPP.16 or AAV9; the promoter is a LSP promoter or an al-antitrypsin promoter; the microRNA targeting sequences target one, two or all three of microRNA 124, mciroRNA 200c and microRNA 1. In some embodiments, the protein of interest is Factor VIII or factor IX.
In some embodiments, the AAV vector is a vector that targets the heart or other muscle; wherein the capsid is AAV.CPP.16; the promoter is a MLC2v promoter or MCK promoter; and the microRNA targeting sequences target one, two or all three of microRNA 122, microRNA 124 and microRNA 200c. In some embodiments, the protein of interest is mini dystrophin or factor IX.
In some embodiments, the AAV vector is a vector that targets the lung, wherein the capsid is AAV.CPP.16; the promoter is an SP-B promoter or SP-C promoter; and the microRNA targeting sequences target microRNA one, two or all three of 122, microRNA 124 and microRNA 1. In some embodiments, the protein of interest is Alpha-1 antitrypsin or an anti-SARS antibody.
Also provided herein are methods of delivering a therapeutic transgene or therapeutic non-coding RNA to a cell of the central nervous system in a subject. The methods include administering an AAV vector as described herein, wherein the AAV vector comprises an AAV capsid, wherein the AAV capsid comprises a peptide insert of up to 21 amino acids, and wherein the peptide insert comprises a targeting peptide as described herein, e.g., 5-7 amino acids of TVSALFK (SEQ ID NO:36); a microRNA targeting sequence, preferably at the 3′UTR; and the therapeutic transgene or therapeutic non-coding RNA. In some embodiments, the peptide insert comprises TVSALFK (SEQ ID NO:36). In some embodiments, the peptide insert comprises TVSALK (SEQ ID NO:32). In some embodiments, the peptide insert consists of TVSALFK (SEQ ID NO: 36). In some embodiments, the peptide insert consists of TVSALK (SEQ ID NO: 32).
In some embodiments, the method comprises delivery to the cortex, cerebellum, hippocampus, substantia nigra, or amygdala. In some embodiments, the method comprises delivery to neurons, astrocytes, glial cells, or cadiomyocytes. In some embodiments, the subject has Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, epilepsy, stroke, brain cancer, spinocerebellar ataxia, Canavan's disease, or brain cancer.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
CNS-targeted gene therapies can use cells inside the CNS as a “bio-factory” to produce potentially therapeutic proteins such as growth factors and antibodies, or anti-cancer therapeutics. Since most cells in the CNS are not evolved to mass-produce foreign transgenes, one approach to expressing transgenes would be to target particular CNS cell types that are capable of expressing transgenes with high efficiency and good tolerance. Transcriptional regulatory elements such as promoters and microRNAs have been employed to achieve cell type specificity for expression of transgenes with varying levels of success. Described herein are combinations of regulatory elements that comprise optimal expression cassettes. These cassettes possess advantageous characteristics for CNS application including 1) high efficiency of transgene expression in the CNS; 2) few side effects on vulnerable cell populations such as the dorsal root ganglion (DRG) cells; 3) reduced exposure of peripheral tissues as a result of inhibiting expression in the periphery such as in the liver.
Tissue specificity of AAV-mediated gene expression can be achieved by applying a combination of vector capsids and expression regulatory elements including promoters and microRNAs.
Described herein are adeno-associated virus (AAV) vectors comprising an AAV capsid, wherein the AAV capsid comprises a peptide insert of up to 21 amino acids, and wherein the peptide insert comprises a targeting peptide as described herein, e.g., 5-7 amino acids of TVSALFK (SEQ ID NO:36), and a microRNA targeting sequence, preferably at the 3′UTR. Also provided herein are AAV-based expression cassettes designed for tissue targeted, e.g., CNS, gene therapy. The cassettes can contain a promoter, followed by a secretory signal peptide sequence, the coding sequence for a therapeutic protein of interest, an optional Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), a polyA signal sequence, and finally a microRNA targeting sequence at the 3′ UTR. The microRNA targeting sequence reduces expression in non-target tissues. See, e.g., Xie et al., Mol Ther. 2011 March; 19(3):526-35.
A preferred viral vector system useful for delivery of nucleic acids in the present methods is the adeno-associated virus (AAV). AAV is a tiny non-enveloped virus having a 25 nm capsid. No disease is known or has been shown to be associated with the wild type virus. AAV has a single-stranded DNA (ssDNA) genome. AAV has been shown to exhibit long-term episomal transgene expression, and AAV has demonstrated excellent transgene expression in the brain, particularly in neurons. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.7 kb. An AAV vector such as that described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985) can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al., Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51:611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790 (1993). There are numerous alternative AAV variants (over 100 have been cloned), and AAV variants have been identified based on desirable characteristics.
In some embodiments, the AAV include targeting peptides that enhance permeation through the BBB, e.g., when inserted into the capsid of an AAV, e.g., AAV1, AAV2, AAV8, or AAV9, or when conjugated to a biological agent, e.g., an antibody or other large biomolecule, either chemically or via expression as a fusion protein.
In some embodiments, the targeting peptides comprise sequences of at least 5 amino acids. In some embodiments, the amino acid sequence comprises at least 4, e.g., 5, contiguous amino acids of the sequences VPALR (SEQ ID NO:29) or VSALK (SEQ ID NO:30).
In some embodiments, the targeting peptides comprise a sequence of X1 X2 X3 X4 X5, wherein:
In some embodiments, the targeting peptides comprise sequences of at least 6 amino acids. In some embodiments, the amino acid sequence comprises at least 4, e.g., 5 or 6 contiguous amino acids of the sequences TVPALR (SEQ ID NO:31), TVSALK (SEQ ID NO:32), TVPMLK (SEQ ID NO:41) and TVPTLK (SEQ ID NO:41).
In some embodiments, the targeting peptides comprise a sequence of X1 X2 X3 X4 X5 X6, wherein:
In some embodiments, the targeting peptides comprise a sequence of X1 X2 X3 X4 X5 X6, wherein:
In some embodiments, the targeting peptides comprise sequences of at least 7 amino acids. In some embodiments, the amino acid sequence comprises at least 4, e.g., 5, 6, or 7 contiguous amino acids of the sequences FTVSALK (SEQ ID NO:33), LTVSALK (SEQ ID NO:34), TVSALFK (SEQ ID NO:36), TVPALFR (SEQ ID NO:37), TVPMLFK (SEQ ID NO:38) and TVPTLFK (SEQ ID NO:39). In some other embodiments, the targeting peptides comprise a sequence of X1 X2 X3 X4 X5 X6 X7, wherein:
In some embodiments, the targeting peptides comprise a sequence of X1 X2 X3 X4 X5 X6 X7, wherein:
In some embodiments, the targeting peptides comprise a sequence of X1 X2 X3 X4 X5 X6 X7, wherein:
In some embodiments, the targeting peptides comprise a sequence of V[S/p][A/m/t/]L (SEQ ID NO: 103), wherein the upper case letters are preferred at that position. In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]L (SEQ ID NO:104). In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]LK (SEQ ID NO:105). In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]LFK. (SEQ ID NO:106).
In some embodiments, the targeting peptide does not consist of VPALR (SEQ ID NO:29) or VSALK (SEQ ID NO:30).
In some embodiments, the peptide insert comprises TVSALFK (SEQ ID NO: 36). In some embodiments, the peptide insert comprises TVSALK (SEQ ID NO:32). In some embodiments, the peptide insert consists of TVSALFK (SEQ ID NO: 36). In some embodiments, the peptide insert consists of TVSALK (SEQ ID NO: 32).
Specific exemplary amino acid sequences that include the above mentioned 5, 6, or 7-amino acid sequences are listed in Table A.
TVSALK
TVSALFK
TVPMLFK
TVPMLK
VPMLKE
VSALKE
VSALKD
Targeting peptides including reversed sequences can also be used, e.g., KLASVT (SEQ ID NO: 107) and KFLASVT (SEQ ID NO: 108). Preferred AAV for use in the present methods and compositions include AAV.CPP.16 and AAV.CPP.21 (described in WO 2020/014471). Other AAV as known in the art (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8 and variants thereof and others as known in the art or described herein) can also be used. AAV.CPP.16 can be used for targeting CNS, liver, heart/muscle, and/or lung.
Exemplary VP1 protein sequences for AAV9, AAV.CPP.16 and AAV.CPP.21 are provided as below:
In some embodiments, the AAV also has one or more additional mutations that increase delivery to the target tissue, e.g., the CNS, or that reduce off-tissue targeting, e.g., mutations that decrease liver delivery when CNS, heart, or muscle delivery is intended (e.g., as described in Pulicherla et al. (2011) Mol Ther 19:1070-1078); or the addition of other targeting peptides, e.g., as described in Chen et al. (2008) Nat Med 15:1215-1218 or Xu et al., (2005) Virology 341:203-214 or U.S. Pat. Nos. 9,102,949; 9,585,971; and US20170166926. See also Gray and Samulski (2011) “Vector design and considerations for CNS applications,” in Gene Vector Design and Application to Treat Nervous System Disorders ed. Glorioso J., editor. (Washington, DC: Society for Neuroscience) 1-9, available at sfn.org/˜/media/SfN/Documents/Short%20Courses/2011%20Short%20Course%20I/2011_SC1_Gray.ashx.
The virus can also include one or more sequences that promote expression of a transgene, e.g., one or more promoter sequences; enhancer sequences, e.g., 5′ untranslated region (UTR) or a 3′ UTR; a polyadenylation site; and/or insulator sequences. In some embodiments, the promoter is a brain tissue specific promoter, e.g., a neuron-specific or glia-specific promoter. In certain embodiments, the promoter is a promoter of a gene selected to from: neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), MeCP2, adenomatous polyposis coli (APC), ionized calcium-binding adapter molecule 1 (Iba-1), synapsin I (SYN), calcium/calmodulin-dependent protein kinase II, tubulin alpha I, neuron-specific enolase and platelet-derived growth factor beta chain. In some embodiments, the promoter is a pan-cell type promoter, e.g., cytomegalovirus (CMV), beta glucuronidase, (GUSB), ubiquitin C (UBC), or rous sarcoma virus (RSV) promoter. GRP78 or HMGB2 promoters can also be used.
In some embodiments, the promoter drives expression in neuronal cells or glial cells. For example, a promoter that drives expression in glial cells can be a GFAP promoter, gfaABC1D promoter, gfa2 promoter, ALDH1L1 promoter, SLC1A3 promoter, Gjb6 promoter, Mbp promoter, MAG promoter, CBh promoter, F4/80 promoter, CD68 promoter, or CD11B promoter. As another example, a promoter that drives expression in neuronal cells can be a neuronal-specific enolase (NSE) promoter, Synapsin promoter, calcium/calmodulin-dependent protein kinase II promoter, tubulin alpha 1 promoter, platelet-derived growth factor beta chain promoter, parvalbumin promoter, GAD67 promoter, or CCK promoter.
In some embodiments, the promoter is a ubiquitous promoter, optionally major immediate early human cytomegalovirus promoter (MIEhCMV), Chicken R-Actin Promoter (CBA); Human Cytomegalovirus Immediate/Early Gene Promoter and Enhancer (CMV); Chicken β-Actin/Cytomegalovirus Hybrid Promoter (CAG); Rous Sarcoma Virus Long Terminal Repeat Promoter (RSV); SV40 promoter; EF1alpha promoter.
Synthetic promoters are also known, see, e.g, Jüttner et al., Nature Neuroscience volume 22, pages 1345-1356(2019); Morelli et al., J Gen Virol. 1999 March; 80 (Pt 3):571-583; O'Carroll et al., Front Mol Neurosci. 2020; 13: 618020 (review).
The AAV vector can also include an enhancer, such as a CMV Enhancer, mDlx enhancer, or AQP4 enhancer. See, e.g., WO2020168279, Nair et al., iScience. 2020 Mar. 27; 23(3): 100888, Gruh et al., J Gene Med. 2008 January; 10(1): 21-32, Abe et al., FEBS Lett. 2017 December; 591(23): 3906-3915, and Dimidschstein et al., Nat Neurosci. 2016 December; 19(12): 1743-1949.
The AAV vector can also include a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) or alternatives, e.g., as described in PCT/EP2014/072852.
Preferably, the vector includes a polyA signal sequence at the 3′ end of the coding sequence for the transgene. Exemplary polyA signal sequence include human growth hormone (hGH), SV40, bovine growth hormone (bGH), or beta-globin, e.g., rabbit beta-globin (rbGlob).
A number of secretory signal peptide sequences are known in the art, including human signal sequences, examples of which are shown in Table 1 (Table adapted from novoprolabs.com/support/articles/commonly-used-leader-peptide-sequences-for-efficient-secretion-of-a-recombinant-protein-expressed-in-mammalian-cells-201804211337.html).
In some embodiments, another secretory sequence that promotes secretion is used, e.g., as described in von Heijne, J Mol Biol. 1985 Jul. 5; 184(1):99-105; Kober et al., Biotechnol. Bioeng. 2013; 110: 1164-1173; Tsuchiya et al., Nucleic Acids Research Supplenzent No. 3 261-262 (2003).
microRNA Targeting Sequences
The AAVs described herein include one or more, e.g., a plurality, e.g., 2-10, 2-8, 2-5, or 3-5, microRNA targeting sequences, optionally separated by spacer sequence (e.g., of 1-50 nucleotides, e.g., comprising a spacer sequence that is not expected to pair with DNA of the targeting sequences to avoid nonspecific binding of miRNA). The following Table 2 provides exemplary miRNAs and their corresponding targeting sequences.
Mouse
Human
Mouse
Human
Mouse
Human
Mouse
Human
In some embodiments, the AAV includes one, two, or three of a microRNA 122 targeting sequence (miR-122T), e.g., targeting the 5p strand, e.g., comprising CAAACACCATTGTCACACTCCA (SEQ ID NO: 21); a microRNA 124 targeting sequence (miR-124T), e.g., targeting the 5p strand, e.g., comprising ATCAAGGTCCGCTGTGAACACG (SEQ ID NO: 20); a microRNA 200c targeting sequence (miR-200cT), e.g., targeting the 5p strand, e.g., CCAAACACTGCTGGGTAAGACG (SEQ ID NO: 22); and/or microRNA 1 targeting sequence (miR-1T), e.g., targeting the 5p strand, e.g., ATGGGCATATAAAGAAGTATGT (SEQ ID NO: 24), at the 3′ UTR.
See also Qiao et al., Gene Ther. 2011 April; 18(4):403-10.
Tissue specificity of AAV-mediated gene expression can be achieved by applying a combination of vector capsids and expression regulatory elements including promoters and microRNAs. For example, for CNS selectivity, a set of miRNAs are used that de-target the liver, lung and/or heart. For muscle selectivity (such as for treating muscle diseases, e.g., Duchenne muscular dystrophy), miRNAs that de-target the liver, lung and the CNS are used. For conditions affecting the CNS and heart, e.g., for applications such as disease Friedreich ataxia, miRNAs that de-target the liver and the lung are used. Exemplary combinations are shown in Table 3. Additional exemplary transgenes are provided in Table B.
In some embodiments, the disclosure provides an AAV vector comprising an AAV capsid comprising a targeting peptide as described herein, e.g., 5-7 amino acids of TVSALFK (SEQ ID NO:36), and an expression cassette comprising a coding sequence for a protein of interest, a therapeutic transgene or non-coding RNA (e.g., as shown in Table B or Table 3), and at least one miR-9 targeting sequence as described herein. In some embodiments, the peptide insert comprises TVSALFK (SEQ ID NO:36). In some embodiments, the peptide insert comprises TVSALK (SEQ ID NO:32). In some embodiments, the peptide insert consists of TVSALFK (SEQ ID NO: 36). In some embodiments, the peptide insert consists of TVSALK (SEQ ID NO: 32). In some embodiments, the targeting peptides comprise a sequence of V[S/p][A/m/t/]L (SEQ ID NO:103), wherein the upper case letters are preferred at that position. In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]L (SEQ ID NO:104). In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]LK (SEQ ID NO:105). In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]LFK. (SEQ ID NO: 106). In some embodiments, the expression cassette comprises a CNS-specific promoter, e.g., a GFAP promoter or Syn promoter. In some embodiments, the coding sequence encodes GDNF, BDNF, AADC, Tau antibody, APP antibody, miRNA targeting HTT, shRNA targeting SOD, IFN-beta, Neuropeptide Y, IGF-1, osteopontin, HSV.TK1, PD-1/PD-L1 antibody, RNAi targeting ataxin, ASPA, ARSA, PSAP, MeCP2, CNTF, ATP7B, or NGF.
In some embodiments, the disclosure provides an AAV vector comprising an AAV capsid comprising a targeting peptide as described herein, e.g., 5-7 amino acids of TVSALFK (SEQ ID NO:36), and an expression cassette comprising a coding sequence for protein of interest, a therapeutic transgene or non-coding RNA (e.g., as shown in Table B or Table 3), and at least one miR-124a targeting sequence as described herein. In some embodiments, the peptide insert comprises TVSALFK (SEQ ID NO:36). In some embodiments, the peptide insert comprises TVSALK (SEQ ID NO:32). In some embodiments, the peptide insert consists of TVSALFK (SEQ ID NO: 36). In some embodiments, the peptide insert consists of TVSALK (SEQ ID NO: 32). In some embodiments, the targeting peptides comprise a sequence of V[S/p][A/m/t/]L (SEQ ID NO:103), wherein the upper case letters are preferred at that position. In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]L (SEQ ID NO:104). In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]LK (SEQ ID NO:105). In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]LFK. (SEQ ID NO: 106). In some embodiments, the expression cassette comprises a CNS-specific promoter, e.g., a GFAP promoter or Syn promoter. In some embodiments, the coding sequence encodes GDNF, BDNF, AADC, Tau antibody, APP antibody, miRNA targeting HTT, shRNA targeting SOD, IFN-beta, Neuropeptide Y, IGF-1, osteopontin, HSV.TK1, PD-1/PD-L1 antibody, RNAi targeting ataxin, ASPA, ARSA, PSAP, MeCP2, CNTF, ATP7B, or NGF.
In some embodiments, the disclosure provides an AAV vector comprising an AAV capsid comprising a targeting peptide as described herein, e.g., 5-7 amino acids of TVSALFK (SEQ ID NO:36), and an expression cassette comprising a coding sequence for a protein of interest, a therapeutic transgene or non-coding RNA (e.g., as shown in Table B or Table 3), and at least one miR-122 targeting sequence as described herein. In some embodiments, the peptide insert comprises TVSALFK (SEQ ID NO:36). In some embodiments, the peptide insert comprises TVSALK (SEQ ID NO:32). In some embodiments, the peptide insert consists of TVSALFK (SEQ ID NO: 36). In some embodiments, the peptide insert consists of TVSALK (SEQ ID NO: 32). In some embodiments, the targeting peptides comprise a sequence of V[S/p][A/m/t/]L (SEQ ID NO:103), wherein the upper case letters are preferred at that position. In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]L (SEQ ID NO:104). In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]LK (SEQ ID NO:105). In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]LFK. (SEQ ID NO: 106). In some embodiments, the expression cassette comprises a liver-specific promoter, e.g., a LSP promoter or al-antitrypsin promoter. In some embodiments, the coding sequence encodes ATP7B, UGT1A1, Factor VIII or factor IX.
In some embodiments, the disclosure provides an AAV vector comprising an AAV capsid comprising a targeting peptide as described herein, e.g., 5-7 amino acids of TVSALFK (SEQ ID NO:36), and an expression cassette comprising a coding sequence for protein of interest, a therapeutic transgene or non-coding RNA (e.g., as shown in Table B or Table 3), and at least one miR-1 targeting sequence as described herein. In some embodiments, the peptide insert comprises TVSALFK (SEQ ID NO:36). In some embodiments, the peptide insert comprises TVSALK (SEQ ID NO:32). In some embodiments, the peptide insert consists of TVSALFK (SEQ ID NO: 36). In some embodiments, the peptide insert consists of TVSALK (SEQ ID NO: 32). In some embodiments, the targeting peptides comprise a sequence of V[S/p][A/m/t/]L (SEQ ID NO:103), wherein the upper case letters are preferred at that position. In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]L (SEQ ID NO:104). In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]LK (SEQ ID NO:105). In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]LFK. (SEQ ID NO: 106). In some embodiments, the expression cassette comprises a heart and/or muscle-specific promoter, e.g., a MLC2v promoter or MCK promoter. In some embodiments, the coding sequence encodes SMN1, Frataxin, MTM1, GAA, TAZ, dystrophin, Mini dystrophin or factor IX.
In some embodiments, the disclosure provides an AAV vector comprising an AAV capsid comprising a targeting peptide as described herein, e.g., 5-7 amino acids of TVSALFK (SEQ ID NO:36), and an expression cassette comprising a coding sequence for protein of interest, a therapeutic transgene or non-coding RNA (e.g., as shown in Table B or Table 3), and at least one miR-208 targeting sequence as described herein. In some embodiments, the peptide insert comprises TVSALFK (SEQ ID NO:36). In some embodiments, the peptide insert comprises TVSALK (SEQ ID NO:32). In some embodiments, the peptide insert consists of TVSALFK (SEQ ID NO: 36). In some embodiments, the peptide insert consists of TVSALK (SEQ ID NO: 32). In some embodiments, the targeting peptides comprise a sequence of V[S/p][A/m/t/]L (SEQ ID NO:103), wherein the upper case letters are preferred at that position. In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]L (SEQ ID NO:104). In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]LK (SEQ ID NO:105). In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]LFK. (SEQ ID NO: 106). In some embodiments, the expression cassette comprises a heart and/or muscle-specific promoter, e.g., a MILC2v promoter or MCK promoter. In some embodiments, the coding sequence encodes SMN1, Frataxin, MTM1, GAA, TAZ, dystrophin, mini dystrophin or factor IX.
In some embodiments, the disclosure provides an AAV vector comprising an AAV capsid comprising a targeting peptide as described herein, e.g., 5-7 amino acids of TVSALFK (SEQ ID NO:36), and an expression cassette comprising a coding sequence for protein of interest as described herein, a therapeutic transgene or non-coding RNA (e.g., as shown in Table B or Table 3), and at least one miR-200C targeting sequence. In some embodiments, the peptide insert comprises TVSALFK (SEQ ID NO:36). In some embodiments, the peptide insert comprises TVSALK (SEQ ID NO:32). In some embodiments, the peptide insert consists of TVSALFK (SEQ ID NO: 36). In some embodiments, the peptide insert consists of TVSALK (SEQ ID NO: 32). In some embodiments, the targeting peptides comprise a sequence of V[S/p][A/m/t/]L (SEQ ID NO:103), wherein the upper case letters are preferred at that position. In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]L (SEQ ID NO:104). In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]LK (SEQ ID NO:105). In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]LFK. (SEQ ID NO:106). In some embodiments, the expression cassette comprises a lung-specific promoter, e.g., a SP-B promoter or SP-C promoter. In some embodiments, the coding sequence encodes Alpha-1 antitrypsin or a therapeutic antibody, e.g., a SARS/COVID antibody.
For example, in some embodiments the cassette comprises a combination of the GFAP promoter and the microRNA122T. The GFAP promoter allows astrocyte-specific transgene expression in the CNS. This strategy diminishes expression of transgenes in the DRG cells and potentially avoids DRG toxicity that is reported to exhibit frequently after AAV-mediated CNS gene delivery. The microRNA122T at the 3′ UTR allows inhibition of expression in the liver since the expression of microRNA 122 is liver-specific. The present data shows that this liver-de-targeting element did not interfere with transgene expression in the CNS.
In some embodiments, the AAV also includes a transgene sequence (i.e., a heterologous sequence), e.g., a transgene encoding a therapeutic agent, e.g., as described herein or as known in the art, or a reporter protein, e.g., a fluorescent protein, an enzyme that catalyzes a reaction yielding a detectable product, or a cell surface antigen. The transgene is preferably linked to regulatory sequences that promote/drive expression of the transgene in the target tissue.
Exemplary transgenes for use as therapeutics include toxins or suicide proteins, which are particularly useful in treating cancers.
Exemplary toxins include diphtheria toxin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), or TNF-α.
Exemplary suicide proteins include herpes simplex virus thymidine kinase (HSVTK), bacterial or fungal cytosine deaminase (CD), carboxypeptidase G2 (CPG2), nitroreductase (NTR), Cytochrome P450 (CYP), purine nucleoside phosphorylase (PNP), horseradish peroxidase (HRP), or carboxylesterase (CE). Such suicide proteins can be used gene directed enzyme prodrug therapy (GDEPT), which combines passive, active, and transcriptional targeting strategies to provide anticancer activity. GDEPT is a two-step process whereby the cells are first transduced by a gene coding for a non-toxic enzyme (suicide gene) followed by administration of a non-toxic prodrug; see, e.g., Karjoo et al., Adv Drug Deliv Rev. 2016 Apr. 1; 99(Pt A): 113-128.
Other therapeutic transgenes include neuronal apoptosis inhibitory protein (NAIP), nerve growth factor (NGF), glial-derived growth factor (GDNF), brain-derived growth factor (BDNF), ciliary neurotrophic factor (CNTF), tyrosine hydroxlase (TH), GTP-cyclohydrolase (GTPCH), amino acid decorboxylase (AADC), aspartoacylase (ASPA), blood factors, such as β-globin, hemoglobin, tissue plasminogen activator, and coagulation factors; colony stimulating factors (CSF); interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, etc.; growth factors, such as keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factor (FGF, such as basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGFs), bone morphogenetic protein (BMP), epidermal growth factor (EGF), growth differentiation factor-9 (GDF-9), hepatoma derived growth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), and the like; soluble receptors, such as soluble TNF-α receptors, soluble VEGF receptors, soluble interleukin receptors (e.g., soluble IL-1 receptors and soluble type II IL-1 receptors), soluble gamma/delta T cell receptors, ligand-binding fragments of a soluble receptor, and the like; enzymes, such as α-glucosidase, imiglucarase, β-glucocerebrosidase, and alglucerase; enzyme activators, such as tissue plasminogen activator; chemokines, such as IP-10, monokine induced by interferon-gamma (Mig), Groa/IL-8, RANTES, MIP-1α, MIP-1β, MCP-1, PF-4, and the like; angiogenic agents, such as vascular endothelial growth factors (VEGFs, e.g., VEGF121, VEGF165, VEGF-C, VEGF-2), transforming growth factor-beta, basic fibroblast growth factor, glioma-derived growth factor, angiogenin, angiogenin-2; and the like; anti-angiogenic agents, such as a soluble VEGF receptor; protein vaccine; neuroactive peptides, such as nerve growth factor (NGF), bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, warfarin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagons, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, a sleep peptide, and the like; thrombolytic agents; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein; follicle stimulating hormone (FSH); human alpha-1 antitryp sin; leukemia inhibitory factor (LIF); transforming growth factors (TGFs); tissue factors, luteinizing hormone; macrophage activating factors; tumor necrosis factor (TNF); neutrophil chemotactic factor (NCF); nerve growth factor; tissue inhibitors of metalloproteinases; vasoactive intestinal peptide; angiogenin; angiotropin; fibrin; hirudin; IL-1 receptor antagonists; and the like. Some other examples of protein of interest include ciliary neurotrophic factor (CNTF); neurotrophins 3 and 4/5 (NT-3 and 4/5); glial cell derived neurotrophic factor (GDNF); aromatic amino acid decarboxylase (AADC); hemophilia related clotting proteins, such as Factor VIII, Factor IX, Factor X; dystrophin or nini-dystrophin; lysosomal acid lipase; phenylalanine hydroxylase (PAH); glycogen storage disease-related enzymes, such as glucose-6-phosphatase, acid maltase, glycogen debranching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase (e.g., PHKA2), glucose transporter (e.g., GLUT2), aldolase A, β-enolase, and glycogen synthase; lysosomal enzymes (e.g., beta-N-acetylhexosaminidase A); and any variants thereof.
The transgene can encode an antibody, e.g., an immune checkpoint inhibitory antibody, e.g., to PD-L1, PD-1, CTLA-4 (Cytotoxic T-Lymphocyte-Associated Protein-4; CD152); LAG-3 (Lymphocyte Activation Gene 3; CD223); TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3; HAVCR2); TIGIT (T-cell Immunoreceptor with Ig and ITIM domains); B7-H3 (CD276); VSIR (V-set immunoregulatory receptor, aka VISTA, B7H5, C10orf54); BTLA 30 (B- and T-Lymphocyte Attenuator, CD272); GARP (Glycoprotein A Repetitions; Predominant; PVRIG (PVR related immunoglobulin domain containing); or VTCN1 (Vset domain containing T cell activation inhibitor 1, aka B7-H4).
Other transgenes can include small or inhibitory nucleic acids that alter/reduce expression of a target gene, e.g., siRNA, shRNA, miRNA, antisense oligos, or long non-coding RNAs that alter gene expression (see, e.g., WO2012087983 and US20140142160), or CRISPR Cas9/casI2a and guide RNAs.
The methods and compositions described herein can be used to express a transgene in a tissue-specific manner, e.g., to the central nervous system (brain), heart, muscle, or dorsal root ganglion or spinal cord (peripheral nervous system). In some embodiments, the methods include systemic delivery of the compositions described herein. In some embodiments, the methods include delivery to specific brain regions, e.g., cortex, cerebellum, hippocampus, substantia nigra, or amygdala. In some embodiments, the methods include delivery to neurons, astrocytes, and/or glial cells.
In some embodiments, the methods and compositions, e.g., AAVs, are used to deliver a nucleic acid sequence to a subject who has a disease, e.g., a disease of the CNS; see, e.g., U.S. Pat. Nos. 9,102,949; 9,585,971; and US20170166926. In some embodiments, the subject has a condition listed in Table B; in some embodiments, the vectors are used to deliver a therapeutic agent listed in Table B for treating the corresponding disease listed in Table B. The therapeutic agent can be delivered as a nucleic acid, e.g. via a viral vector, wherein the nucleic acid encodes a therapeutic protein or other nucleic acid such as an antisense oligo, siRNA, shRNA, and so on; or as a fusion protein/complex with a targeting peptide as described herein.
In some embodiments, the methods and compositions, e.g., AAVs, are used to deliver a nucleic acid sequence encoding a transgene that includes a toxin or suicide gene to a subject who has brain cancer. Brain cancers include gliomas (e.g., glioblastoma multiforme (GBM)), metastases (e.g., from lung, breast, melanoma, or colon cancer), meningiomas, pituitary adenomas, and acoustic neuromas. Thus the methods can include systemically, e.g., intravenously, administering an AAV.CPP.16 as described herein, encoding a toxin or suicide protein to a subject who has been diagnosed with brain cancer. In some embodiments, where the therapeutic protein is a suicide protein, the methods further include contact administering a nontoxic prodrug that is a substrate for the suicide protein, wherein action of the suicide protein on the nontoxic prodrug results in production of a toxic metabolite that induces cell death. Examples include wherein the suicide protein is herpes simplex virus thymidine kinase (HSVTK) and the nontoxic prodrug is ganciclovir (GCV); the suicide protein is cytosine deaminase (CD) and the nontoxic prodrug is 5-flourouracil (5-FU); the suicide protein is carboxypeptidase G2 (CPG2) and the nontoxic prodrug is nitrogen mustard (NM) or a derivate thereof such as ZD2767P or CMDA (4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamicacid); the suicide protein is nitroreductase (NTR) and the nontoxic prodrug is CB1954 or an analog thereof; the suicide protein is Cytochrome P450 (CYP) and the nontoxic prodrug is an oxazaphosphorine drug such as cyclophosphamide (CPA) and ifosfomide (IFO); the suicide protein is purine nucleoside phosphorylase (PNP) and the nontoxic prodrug is 6-Methylpurine Deoxyriboside or an analog thereof, e.g., fludarabine phosphate (F-araAMP) or 2-fluoro-2-deoxyadenosine (F-dAdo); the suicide gene is horseradish peroxidase (HRP) and the nontoxic prodrug is indole-3-acetic acid (HRP/IAA); or the suicide protein is carboxylesterase (CE) and the nontoxic prodrug is irinotecan. See, e.g., Karjoo et al., Adv Drug Deliv Rev. 2016 Apr. 1; 99(Pt A): 113-128.
In some embodiments, the AAV is targeted to glial cells, e.g., cancerous glial cells in a subject, e.g., the cancer is glioblastoma.
In some embodiments, the methods also include co-administering a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is a toxin or cytotoxic drug, including but not limited to temozolamide, lomustine, or a combination thereof. See, e.g., Herrlinger et al., Lancet. 2019 Feb. 16; 393(10172):678-688. The methods can also include administering radiation, surgical resection, or both.
The methods described herein include the use of pharmaceutical compositions comprising or consisting of AAVs as described herein an active ingredient.
Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intraarterial, subcutaneous, intraperitoneal intramuscular or injection or infusion administration. Delivery can thus be systemic or localized.
Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
In one embodiment, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
As illustrated in
To demonstrate of proof-of-concept, a liver-detargeting, AAV.CPP.16-based vector was developed for CNS cancer gene therapy. As illustrated in
To demonstrate the function of such expression cassette, AAVs.CPP.16 containing such cassette (AAV.CPP.16-GFAP-RFP-miR122T) were produced along with AAV.CPP.16-GFAP-RFP. As shown in
To further test both vectors in vivo, AAVs.CPP.16-GFAP-RFP-miR122T and AAVs.CPP.16-GFAP-RFP were intravenously injected into adult BALB/c mice at a dose of 1e12 vg per animal. 3 weeks later, animals were processed and brains, as well as other peripheral tissues, were examined for RFP expression. As shown in
Significant difference of transduction was found in the liver between the two AAV vectors. As shown in
As shown in
Herpes simplex virus thymidine kinase 1 (i.e., HSV TK1) is a “suicide” gene that can be applied for killing tumor cells. As illustrated in
It was found that when AAVs.CPP.16-GFAP-TK1-miR122T were intravenously administered into adult mice bearing GL261 tumor cells, TK1 expression was observed in the brain while no such expression was observed in the liver, as shown in
It was further found that combining AAVs.CPP.16-GFAP-TK1-miR122T with GCV administration dramatically reduced the glioma tumor size in the brain compared with shame control treatment, as shown in
In this example, AAV.CPP.16-GFAP-TK1-miR122T was intravenously administered to GL261 glioblastoma tumor-bearing mice using the experimental timeline depicted in
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Patent Application No. 63/213,045, filed Jun. 21, 2021. The entire contents of the foregoing are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/073051 | 6/21/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63213045 | Jun 2021 | US |
