Compositions for Treatment of Vascular Disease

Abstract

Provided are various embodiments relating to compositions and methods for treating vascular disease, including core NOX1 promoters and variants thereof for regulating expression of transgenes in response to vascular pathology and allowing for increased transgene loading capacity. Also provided are variant FOXP polypeptides having a zinc finger and leucine zipper region of a different FOXP polypeptide. Further provided are vectors comprising the core NOX1 promoters and/or a coding sequence for variant FOXP polypeptides described herein and optionally coding sequence(s) for one or more additional therapeutic polypeptide(s), such as IL10, for treating inflammation-associated diseases, such as vascular disease. Also provided is a screening model for testing therapeutic agents capable of treating established and ongoing atherosclerotic pathology.

What is claimed is: 
     
         1 . An isolated nucleic acid molecule comprising a NOX1 core promoter and a heterologous nucleic acid molecule, wherein the NOX1 core promoter comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3. 
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein the NOX1 core promoter is less than 600 nucleotides, less than 550 nucleotides, less than 500 nucleotides, less than 480 nucleotides, or less than 470 nucleotides. 
     
     
         3 . The isolated nucleic acid molecule of any one of the preceding claims, comprising at least one heterologous NFκB binding site. 
     
     
         4 . The isolated nucleic acid molecule of  claim 3 , wherein the at least one heterologous NFκB binding site comprises the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20. 
     
     
         5 . The isolated nucleic acid molecule of  claim 3  or  claim 4 , wherein the at least one NFκB binding site comprises the nucleotide sequence of SEQ ID NO: 5. 
     
     
         6 . The isolated nucleic acid molecule of any one of the preceding claims, comprising at least two, at least three, at least four, or at least five heterologous NFκB binding sites. 
     
     
         7 . The isolated nucleic acid molecule of any one of the preceding claims, comprising at least one heterologous Oct1 binding site. 
     
     
         8 . The isolated nucleic acid molecule of  claim 7 , wherein the at least one Oct1 binding site comprises the nucleotide sequence of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, and/or SEQ ID NO: 21. 
     
     
         9 . The isolated nucleic acid molecule of  claim 7  or  claim 8 , wherein the at least one Oct1 binding site comprises the nucleotide sequence of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, and/or SEQ ID NO: 21. 
     
     
         10 . The isolated nucleic acid molecule of any one of  claim 7  to  claim 9 , wherein the at least one Oct1 binding site comprises the nucleotide sequence of SEQ ID NO: 21. 
     
     
         11 . The isolated nucleic acid molecule of any one of the preceding claims, comprising at least two, at least three, at least four, or at least five Oct1 binding sites. 
     
     
         12 . An isolated nucleic acid molecule comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 4. 
     
     
         13 . The isolated nucleic acid molecule of any one of the preceding claims, comprising the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. 
     
     
         14 . An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2 and a heterologous nucleotide sequence. 
     
     
         15 . An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 3. 
     
     
         16 . An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 4. 
     
     
         17 . The isolated nucleic acid molecule of  claim 15  or  claim 16 , comprising a heterologous nucleic acid molecule. 
     
     
         18 . The isolated nucleic acid molecule of any one of the preceding claims, wherein the heterologous nucleic acid molecule comprises a heterologous nucleotide sequence encoding an anti-inflammatory molecule or a reporter protein. 
     
     
         19 . The isolated nucleic acid molecule of  claim 18 , wherein the anti-inflammatory molecule is a wildtype or variant FOXP3 polypeptide and/or IL10. 
     
     
         20 . The isolated nucleic acid molecule of  claim 19 , wherein the wildtype or variant FOXP3 polypeptide comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 28. 
     
     
         21 . An isolated variant FOXP polypeptide comprising the amino acid sequence of a first FOXP polypeptide, wherein a zinc finger and leucine zipper region of the first FOXP polypeptide has been replaced with a zinc finger and leucine zipper region of a second FOXP polypeptide. 
     
     
         22 . The isolated variant FOXP polypeptide of  claim 21 , wherein:
 a) the first FOXP polypeptide is a FOXP3 polypeptide and the second FOXP polypeptide is a FOXP1 polypeptide, a FOXP2 polypeptide, or a FOXP4 polypeptide;   b) the first FOXP polypeptide is a FOXP1 polypeptide and the second FOXP polypeptide is a FOXP2 polypeptide, a FOXP3 polypeptide, or a FOXP4 polypeptide;   c) the first FOXP polypeptide is a FOXP2 polypeptide and the second FOXP polypeptide is a FOXP1 polypeptide, a FOXP3 polypeptide, or a FOXP4 polypeptide; or   d) the first FOXP polypeptide is a FOX4 polypeptide and the second FOXP polypeptide is a FOXP1 polypeptide, a FOXP2 polypeptide, or a FOXP3 polypeptide.   
     
     
         23 . The isolated variant FOXP polypeptide of  claim 21  or  claim 22 , wherein the first FOXP polypeptide is a FOXP3 polypeptide and the second FOXP polypeptide is a FOXP1 polypeptide. 
     
     
         24 . The isolated variant FOXP polypeptide of any one of  claims 21  to  23 , wherein the zinc finger and leucine zipper region of the second FOXP polypeptide comprises the amino acid sequence of SEQ ID NO: 27. 
     
     
         25 . The isolated variant FOXP polypeptide of any one of  claims 21  to  24 , comprising an amino acid sequence having at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 28. 
     
     
         26 . The isolated variant FOXP polypeptide of any one of  claims 21  to  25 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 28. 
     
     
         27 . An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 28. 
     
     
         28 . An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: 29 or SEQ ID NO: 34. 
     
     
         29 . An isolated nucleic acid comprising a nucleic acid sequence encoding the variant FOXP polypeptide of any one of  claims 21  to  27 . 
     
     
         30 . An isolated contiguous nucleic acid comprising a first nucleic acid sequence encoding a first therapeutic polypeptide and a second nucleic acid sequence encoding a second therapeutic polypeptide, wherein the first therapeutic polypeptide comprises the variant FOXP polypeptide of any one of  claims 21  to  27 . 
     
     
         31 . The isolated nucleic acid of  claim 30 , wherein the second nucleic acid sequence is downstream of the first nucleic acid sequence. 
     
     
         32 . The isolated nucleotide of  claim 30 , wherein the first nucleic acid sequence is downstream of the second nucleic acid sequence. 
     
     
         33 . The isolated nucleic acid of any one of  claims 30  to  32 , wherein the second therapeutic polypeptide is a signaling protein, such as a cytokine, a growth factor, or a chemokine. 
     
     
         34 . The isolated nucleic acid of any one of  claims 30  to  33 , wherein the second therapeutic polypeptide is selected from the group consisting of IL6, IL10, IL11, IL13, TGFβ, IL4, IL1ra, soluble TNF receptor sp55, soluble TNF receptor sp75, sIL-1RII, mIL-1RII, and IL18BP. 
     
     
         35 . The isolated nucleic acid of any one of  claims 30  to  33 , wherein the second therapeutic polypeptide is an IL10 polypeptide. 
     
     
         36 . The isolated nucleic acid of any one of  claims 30  to  35 , wherein the second therapeutic polypeptide comprises the amino acid sequence of SEQ ID NO: 31. 
     
     
         37 . The isolated nucleic acid of any one of  claims 30  to  36 , wherein the first nucleic acid sequence and/or the second nucleic acid sequence is operatively linked to a first promoter. 
     
     
         38 . The isolated nucleic acid of any one of  claims 30  to  37 , wherein the second nucleic acid sequence is operatively linked to a second promoter. 
     
     
         39 . The isolated nucleic acid of  claim 37  or  claim 38 , wherein the first promoter and/or the second promoter is a constitutive promoter. 
     
     
         40 . The isolated nucleic acid of any one of  claims 37  to  39 , wherein the first promoter and/or the second promoter is a cytomegalovirus (CMV) immediate early promoter, a simian virus 40 (SV40) early promoter, a phosphoglycerate kinase 1 (PGK1) promoter, a human β-actin promoter, or a chicken β-actin promoter and CMV early enhancer. 
     
     
         41 . The isolated nucleic acid of any one of  claims 37  to  40 , wherein the first promoter and/or the second promoter is a regulated promoter. 
     
     
         42 . The isolated nucleic acid of any one of  claims 37  to  41 , wherein the first promoter and/or the second promoter is a tissue-specific promoter or a pathology-specific promoter. 
     
     
         43 . The isolated nucleic acid of any one of  claims 37  to  42 , wherein the first promoter and/or the second promoter is activated by sheer stress and/or dyslipidemia. 
     
     
         44 . The isolated nucleic acid of any one of  claims 37  to  43 , wherein the first promoter and/or the second promoter is activated by Angiotensin-2 (Ang II), Lipopolysaccharides (LPS), Oxidized Low Density Lipoprotein (Ox-LDL), and/or carbamylated LDL. 
     
     
         45 . The isolated nucleic acid of any one of  claims 37  to  44 , wherein the first promoter and/or the second promoter comprises the nucleic acid of any one of  claims 1  to  17 . 
     
     
         46 . The isolated nucleic acid of any one of  claims 28  to  45 , comprising a TATA box between the first nucleic acid sequence and the second nucleic acid sequence. 
     
     
         47 . The isolated nucleic acid of any one of  claims 28  to  46 , comprising at least one polyadenylation sequence. 
     
     
         48 . The isolated nucleic acid of any one of  claims 28  to  47 , comprising adeno-associated virus (AAV) inverted terminal repeats. 
     
     
         49 . A vector comprising the nucleic acid of any one of  claims 1  to  20  or any one of  claims 28  to  48 . 
     
     
         50 . The vector of  claim 49 , wherein the vector is a virus vector. 
     
     
         51 . The vector of  claim 49  or  claim 50 , wherein the vector is an adeno-associated virus (AAV) vector, an adenovirus vector, a retrovirus vector, a herpesvirus vector, or a pox virus vector. 
     
     
         52 . The vector of any one of  claims 49  to  51 , wherein the vector is an adeno-associated virus (AAV) vector. 
     
     
         53 . The vector of any one of  claims 49  to  52 , wherein the vector is an adeno-associated virus (AAV) vector having a capsid serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, and any variant thereof. 
     
     
         54 . The vector of any one of  claims 49  to  53 , further comprising one or more peptides for targeting the vector to a host cell. 
     
     
         55 . A vector comprising one or more peptides for targeting the vector to a host cell, wherein the one or more peptides target LOX-1. 
     
     
         56 . The vector of  claim 54  or  55 , wherein the one or more peptides target LOX-1. 
     
     
         57 . The vector of any one of  claims 54  to  56 , wherein the one or more peptides are 3 to 150 residues long. 
     
     
         58 . The vector of any one of  claims 54  to  57 , wherein the one or more peptides are 3 to 32 residues long. 
     
     
         59 . The vector of any one of  claims 54  to  58 , wherein the one or more peptides are 3, 4, 5, 6, 7, 8, 9, or 10 residues long. 
     
     
         60 . The vector of any one of  claims 54  to  59 , wherein the one or more peptides are 7 residues long. 
     
     
         61 . The vector of any one of  claims 54  to  60 , wherein the one or more peptides comprise an amino acid sequence of any one of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102. 
     
     
         62 . The vector of any one of  claims 54  to  61 , wherein the one or more peptides comprise an amino acid sequence of any one of SEQ ID NO: 74, SEQ ID NO: 59, and SEQ ID NO: 82. 
     
     
         63 . The vector of any one of  claims 54  to  62 , wherein the one or more peptides are incorporated in or associated with a capsid protein of the vector. 
     
     
         64 . The vector of any one of  claims 54  to  63 , wherein the one or more peptides are positioned within an AAV VP2 or VP3 capsid protein. 
     
     
         65 . The vector of any one of  claims 54  to  64 , wherein the one or more peptides are positioned between amino acid residues 587 and 588 of an AAV2 VP3 capsid protein. 
     
     
         66 . The vector of any one of  claims 54  to  64 , wherein the one or more peptides are positioned between amino acid residues 585 and 586 or between amino acid residues 590 and 591 of an AAV8 VP3 capsid protein. 
     
     
         67 . The vector of any one of  claims 54  to  64 , wherein the one or more peptides are positioned at the N terminus of AAV2 VP2 capsid protein. 
     
     
         68 . The vector of any one of  claims 54  to  64 , wherein the one or more peptides are conjugated to the vector. 
     
     
         69 . The vector of any one of  claims 54  to  65 , wherein the one or more peptides are conjugated to a capsid protein of the vector. 
     
     
         70 . A cultured host cell comprising the nucleic acid molecule of any one of  claims 1  to  20  or any one of  claims 28  to  48 , the variant FOXP polypeptide of any one of  claims 21  to  26 , or the vector of any one of  claims 49  to  69 . 
     
     
         71 . A pharmaceutical composition comprising the nucleic acid molecule of any one of  claims 1  to  20  or any one of  claims 28  to  48 , the variant FOXP polypeptide of any one of  claims 21  to  26 , the vector of any one of  claims 49  to  69 , or the cultured host cell of  claim 70 . 
     
     
         72 . A method comprising administering to a subject the nucleic acid molecule of any one of  claims 1  to  20  or any one of  claims 28  to  48 , the variant FOXP polypeptide of any one of  claims 21  to  26 , the vector of any one of  claims 49  to  69 , the cultured host cell of  claim 70 , or the pharmaceutical composition of  claim 71 . 
     
     
         73 . A method of treating a subject comprising administering to the subject the nucleic acid molecule of any one of  claims 1  to  20  or any one of  claims 28  to  48 , the variant FOXP polypeptide of any one of  claims 21  to  26 , the vector of any one of  claims 49  to  69 , the cultured host cell of  claim 70 , or the pharmaceutical composition of  claim 71 . 
     
     
         74 . The method of  claim 72  or  claim 73 , wherein the subject has a vascular disease and/or a cardiovascular disease. 
     
     
         75 . The method of any one of  claims 72  to  74 , wherein the subject has an inflammation-associated disease. 
     
     
         76 . The method of any one of  claims 72  to  75 , wherein the subject has an age-associated disease. 
     
     
         77 . The method of any one of  claims 72  to  76 , wherein the subject has atherosclerosis. 
     
     
         78 . The method of any one of  claims 72  to  77 , wherein the subject has arthritis, such as psoriatic arthritis, rheumatoid arthritis, and/or gouty arthritis. 
     
     
         79 . The method of any one of  claims 72  to  78 , wherein the subject has dementia. 
     
     
         80 . The method of any one of  claims 72  to  79 , wherein the subject has Alzheimer's disease. 
     
     
         81 . The method of any one of  claims 72  to  80 , wherein the subject has asthma. 
     
     
         82 . The method of any one of  claims 72  to  81 , wherein the subject has macular degeneration of the retina. 
     
     
         83 . The method of any one of  claims 72  to  82 , wherein the subject has arterial disease of the aorta, carotid artery disease, coronary artery disease, atherosclerotic cerebrovascular disease, peripheral artery disease, and/or diabetes mellitus. 
     
     
         84 . A method of screening a therapeutic agent comprising:
 a) maintaining a low density lipoprotein receptor knockout (LDLR-KO) or Apo E knockout animal (such as a mouse or rat) on a high cholesterol diet; and   b) administering a therapeutic agent to the animal no earlier than 56 days after beginning the high cholesterol diet.   
     
     
         85 . The method of  claim 84 , wherein the high cholesterol diet comprises at least 0.1%, at least 0.15%, at least 0.2%, at least 0.4%, at least 0.5%, at least 1%, at least 1.25%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, or at least 5% cholesterol. 
     
     
         86 . The method of  claim 84  or  claim 85 , wherein the high cholesterol diet comprises from about 0.1% to about 5% cholesterol. 
     
     
         87 . The method of any one of  claims 84  to  86 , wherein the high cholesterol diet comprises from about 1 to about 15% cocoa butter. 
     
     
         88 . The method of any one of  claims 84  to  87 , wherein the high cholesterol diet comprises about 4% cholesterol and about 10% cocoa butter. 
     
     
         89 . The method of any one of  claims 84  to  88 , wherein the high cholesterol diet further comprises added sugar, added salt, or both added sugar and added salt. 
     
     
         90 . The method of  claim 89 , wherein the added sugar comprises one or more of fructose, high fructose corn syrup, sucrose, and glucose. 
     
     
         91 . The method of any one of  claims 89  to  90 , wherein the added sugar is added to the drinking water. 
     
     
         92 . The method of any one of  claims 89  to  91 , wherein the added sugar equals at least about 20% of energy in the high cholesterol diet. 
     
     
         93 . The method of any one of  claims 89  to  92 , wherein the added sugar equals at least about 25% of energy in the high cholesterol diet. 
     
     
         94 . The method of any one of  claims 89  to  93 , wherein the added sugar equals at least about 60% of energy in the high cholesterol diet. 
     
     
         95 . The method of any one of  claims 89  to  94 , wherein the added salt comprises a sodium-rich chow of about 4% or 8% NaCl, tap water containing about 1% NaCl ad libitum, or both. 
     
     
         96 . The method of any one of  claims 84  to  95 , wherein the method further comprises:
 c) assessing the blood flow velocity, cross-sectional area of the aorta lumen, and/or the aortic wall thickness of the animal by ultrasound imaging. 
 
     
     
         97 . The method of any one of  claims 84  to  96 , wherein the therapeutic agent is administered no earlier than 63 days, no earlier than 70 days, no earlier than 77 days, no earlier than 84 days, no earlier than 91 days, or no earlier than 98 days after beginning the high cholesterol diet. 
     
     
         98 . The method of any one of  claims 84  to  97 , wherein the therapeutic agent is the nucleic acid of any one of  claims 1  to  20  or any one of  claims 28  to  48 , the variant FOXP polypeptide of any one of  claims 21  to  26 , the vector of any one of  claims 49  to  69 , or the pharmaceutical composition of  claim 71 . 
     
     
         99 . The method of any one of  claims 72  to  98 , wherein the nucleic acid, the variant FOXP polypeptide, the vector, the cultured host cell, the pharmaceutical composition, or the therapeutic agent is administered to the subject or the animal once. 
     
     
         100 . The method of any one of  claims 72  to  99 , wherein the nucleic acid, the variant FOXP polypeptide, the vector, the cultured host cell, the pharmaceutical composition, or the therapeutic agent is administered to the subject or the animal every other day, weekly, or monthly. 
     
     
         101 . The method of any one of  claims 72  to  100 , wherein the variant FOXP polypeptide, the vector, the cultured host cell, the pharmaceutical composition, or the therapeutic agent is administered to the subject or the animal parenterally. 
     
     
         102 . The method of any one of  claims 72  to  101 , wherein the nucleic acid, the variant FOXP polypeptide, the vector, the cultured host cell, the pharmaceutical composition, or the therapeutic agent is administered to the subject or the animal via intravenous injection, arterial injection, intramuscular injection, injection into a section of ligated artery or vein, intraperitoneal injection, subcutaneous injection, intrathecal injection, and/or injection or inhalation to the lung. 
     
     
         103 . The method of any one of  claims 72  to  102 , wherein the nucleic acid, the variant FOXP polypeptide, the vector, the cultured host cell, the pharmaceutical composition, or the therapeutic agent is administered to the subject or the animal via gel, cream, and/or suspension applied to one or more locations of interest. 
     
     
         104 . The method of any one of  claims 72  to  103 , wherein the nucleic acid, the variant FOXP polypeptide, the vector, the cultured host cell, the pharmaceutical composition, or the therapeutic agent is administered to the subject or the animal via a combination of more than one method. 
     
     
         105 . The method of any one of  claims 72  to  104 , wherein a therapeutically effective amount of the nucleic acid, the variant FOXP polypeptide, the vector, the cultured host cell, the pharmaceutical composition, or the therapeutic agent is administered to the subject or the animal. 
     
     
         106 . The method of any one of  claims 72  to  105 , wherein the vector is administered to the subject or the animal at a dose of about 1×10 10  encapsidated genomes, about 1×10 11  encapsidated genomes, about 1×10 12  encapsidated genomes, about 1×10 13  encapsidated genomes, about 1×10 10  to about 1×10 13  encapsidated genomes, about 1×10 10  to about 1×10 12  encapsidated genomes, about 1×10 11  to about 1×10 12  encapsidated genomes, or about 1×10 10  to about 1×10 11  encapsidated genomes.