Compositions of DNA Molecules, Methods of Making Therefor, and Methods of Use Thereof

Abstract

Provided herein are double strand DNA molecules comprising inverted repeats, expression cassette and one or more restriction sites for nicking endonucleases, the methods of use thereof, and the methods of making therefor.

1 . A double-stranded DNA molecule comprising in 5′ to 3′ direction of the top strand:
 a. a first inverted repeat, wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in:
 i. a top strand 5′ single strand DNA overhang comprising the first inverted repeat or a fragment thereof upon separation of the top from the bottom strand of the first inverted repeat; or 
 ii. a bottom strand 3′ single strand DNA overhang comprising the first inverted repeat or a fragment thereof upon separation of the top from the bottom strand of the first inverted repeat; and 
 
 b. an expression cassette; and 
 c. a second inverted repeat, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in:
 i. a top strand 3′ single strand DNA overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand of the second inverted repeat; or 
 ii. a bottom strand 5′ single strand DNA overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat. 
 
 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . The double-stranded DNA molecule of  claim 1 , wherein the double-stranded DNA molecule is an isolated DNA molecule. 
     
     
         6 . The double-stranded DNA molecule of  claim 1 , wherein the first, second, third, and fourth restriction sites for nicking endonuclease are all restriction sites for the same nicking endonuclease. 
     
     
         7 . The double-stranded DNA molecule of  claim 1 , wherein the first and the second inverted repeats are the same. 
     
     
         8 . The double-stranded DNA molecule of  claim 1 , wherein the first and/or the second inverted repeat is an ITR of a parvovirus. 
     
     
         9 . The double-stranded DNA molecule of  claim 1 , wherein the first and/or the second inverted repeat is a modified ITR of a parvovirus. 
     
     
         10 . The double-stranded DNA molecule of  claim 8 , wherein the parvovirus is a Dependoparvovirus, a Bocaparvovirus, an Erythroparvovirus, a Protoparvovirus, or a Tetraparvovirus. 
     
     
         11 . The double-stranded DNA molecule of  claim 9  wherein the nucleotide sequence of the modified ITR is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% identical to the ITR of the parvovirus. 
     
     
         12 . The double-stranded DNA molecule of  claim 1 , wherein the
 a. the first nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5′ nucleotide of the ITR closing base pair of the first inverted repeat;   b. the second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3′ nucleotide of the ITR closing base pair of the first inverted repeat;   c. the third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5′ nucleotide of the ITR closing base pair of the second inverted repeat, and the fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3′ nucleotide of the ITR closing base pair of the second inverted repeat; or   d. the third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3′ nucleotide of the ITR closing base pair of the second inverted repeat, and the fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5′ nucleotide of the ITR closing base pair of the second inverted repeat;
 wherein the first nick and the fourth nick are two resulting nicks on the top strand, with the first nick being the one closer to the 5′ end of the top strand and the fourth nick being the one closer to the 3′ end of the top strand, and wherein the second nick and the third nick are two resulting nicks on the bottom strand, with the third nick being the one closer to the 5′ end of the bottom strand and the second nick being the one closer to the 3′ end of the bottom strand. 
   
     
     
         13 . The double-stranded DNA molecule of  claim 1 , wherein the
 a. the first nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3′ nucleotide of the ITR closing base pair of the first inverted repeat;   b. the second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5′ nucleotide of the ITR closing base pair of the first inverted repeat;   c. the third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3′ nucleotide of the ITR closing base pair of the second inverted repeat, and the fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5′ nucleotide of the ITR closing base pair of the second inverted repeat; or   d. the third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5′ nucleotide of the ITR closing base pair of the second inverted repeat, and the fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3′ nucleotide of the ITR closing base pair of the second inverted repeat;
 wherein the first nick and the fourth nick are two resulting nicks on the top strand, with the first nick being the one closer to the 5′ end of the top strand and the fourth nick being the one closer to the 3′ end of the top strand, and wherein the second nick and the third nick are two resulting nicks on the bottom strand, with the third nick being the one closer to the 5′ end of the bottom strand and the second nick being the one closer to the 3′ end of the bottom strand. 
   
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . The double-stranded DNA molecule of  claim 1 , wherein the double-stranded DNA molecule is a plasmid. 
     
     
         19 . The double-stranded DNA molecule of  claim 18 , wherein the plasmid further comprises a bacterial origin of replication. 
     
     
         20 . The double-stranded DNA molecule of  claim 18 , wherein the plasmid further comprises a restriction enzyme site in the region 5′ to the first inverted repeat and 3′ to the second inverted repeat wherein the restriction enzyme site is not present in any of the first inverted repeat, second inverted repeat, and the region between the first and second inverted repeats. 
     
     
         21 . The double-stranded DNA molecule of  claim 20 , wherein the cleavage with the restriction enzyme results in single strand overhangs that do not anneal at detectable levels under conditions that favor annealing of the first and/or second inverted repeat. 
     
     
         22 . The double-stranded DNA molecule of  claim 20 , wherein the plasmid further comprises an open reading frame encoding the restriction enzyme. 
     
     
         23 . The double-stranded DNA molecule of  claim 22 , wherein expression of the restriction enzyme is under the control of an inducible promoter. 
     
     
         24 . The double-stranded DNA molecule of  claim 18 , wherein the plasmid further comprises a fifth and a sixth restriction site for nicking endonuclease in the region 5′ to the first inverted repeat and 3′ to the second inverted repeat, wherein the fifth and sixth restriction sites for nicking endonuclease are:
 a. on opposite strands; and 
 b. create a break in the double stranded DNA molecule such that the single strand overhangs of the break do not anneal at detectable levels inter- or intramolecularly under conditions that favor annealing of the first and/or second inverted repeat. 
 
     
     
         25 . The double-stranded DNA molecule of  claim 24 , wherein the fifth and the sixth nick are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides apart. 
     
     
         26 . The double-stranded DNA molecule of  claim 24 , wherein the first, second, third, fourth, fifth, and sixth restriction sites for nicking endonuclease are all target sequences for the same nicking endonuclease. 
     
     
         27 . The double-stranded DNA molecule of  claim 1 , wherein one or more of the nicking endonuclease site(s) is a target sequence of an endogenous nicking endonuclease. 
     
     
         28 . The double-stranded DNA molecule of  claim 24 , wherein the plasmid further comprises an open reading frame encoding a nicking endonuclease that recognizes the first, second, third, fourth, fifth, and/or sixth restriction site for nicking endonuclease. 
     
     
         29 . The double-stranded DNA molecule of  claim 28 , wherein expression of the nicking endonuclease is under the control of an inducible promoter. 
     
     
         30 . The double-stranded DNA molecule of  claim 1  wherein the nicking endonuclease that recognizes the first, second, third, and/or fourth restriction site for nicking endonuclease is Nt. BsmAI; Nt. BtsCI; N. ALwl; N. BstNBI; N. BspD6I; Nb. Mva1269I; Nb. BsrDI; Nt. BtsI; Nt. BsaI; Nt. Bpu10I; Nt. BsmBI; Nb. BbvCI; Nt. BbvCI; or Nt. BspQI. 
     
     
         31 . The double-stranded DNA molecule of  claim 24  wherein the nicking endonuclease that recognizes the fifth and sixth restriction site for nicking endonuclease Nt. BsmAI; Nt. BtsCI; N. ALwl; N. BstNBI; N. BspD6I; Nb. Mva1269I; Nb. BsrDI; Nt. BtsI; Nt. BsaI; Nt. Bpu10I; Nt. BsmBI; Nb. BbvCI; Nt. BbvCI; or Nt. BspQI. 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . The double-stranded DNA molecule of  claim 1 , wherein the size of the expression cassette is at least 4 kb, at least 4.5 kb, at least 5 kb, at least 5.5 kb, at least 6 kb, at least 6.5 kb, at least 7 kb, at least 7.5 kb, at least 8 kb, at least 8.5 kb, at least 9 kb, at least 9.5 kb, or at least 10 kb. 
     
     
         37 . A double-stranded DNA molecule comprising in 5′ to 3′ direction of the top strand:
 a. a first hairpinned inverted repeat; 
 b. a nick of the bottom strand or of the top strand; 
 c. an expression cassette; 
 d. a nick of the bottom strand or of the top strand; and 
 e. a second hairpinned inverted repeat. 
 
     
     
         38 . (canceled) 
     
     
         39 . (canceled) 
     
     
         40 . (canceled) 
     
     
         41 . The double-stranded DNA molecule of  claim 37 , which is an isolated DNA molecule. 
     
     
         42 . The double-stranded DNA molecule of  claim 37 , wherein:
 a. the first and/or the second inverted repeat is the ITR of a parvovirus; and/or   b. the first and the second inverted repeats are the same; and/or   c. the first and/or the second inverted repeat is a modified ITR of a parvovirus.   
     
     
         43 . (canceled) 
     
     
         44 . (canceled) 
     
     
         45 . The double-stranded DNA molecule of  claim 42 , wherein the parvovirus is a Dependoparvovirus, a Bocaparvovirus, an Erythroparvovirus, a Protoparvovirus, or a Tetraparvovirus. 
     
     
         46 . The double-stranded DNA molecule of 45, wherein the nucleotide sequence of the modified ITR is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% identical to the ITR of the parvovirus. 
     
     
         47 . (canceled) 
     
     
         48 . (canceled) 
     
     
         49 . (canceled) 
     
     
         50 . (canceled) 
     
     
         51 . The double-stranded DNA molecule of  claim 37 , wherein the size of the expression cassette is at least 4 kb, at least 4.5 kb, at least 5 kb, at least 5.5 kb, at least 6 kb, at least 6.5 kb, at least 7 kb, at least 7.5 kb, at least 8 kb, at least 8.5 kb, at least 9 kb, at least 9.5 kb, or at least 10 kb. 
     
     
         52 . The double-stranded DNA molecule of  claim 37 , wherein the double-stranded DNA molecule is protected from an exonuclease, optionally wherein the exonuclease is RecBCD exonuclease. 
     
     
         53 . The double-stranded DNA molecule of  claim 37 , wherein the double-stranded DNA molecule lacks at least one functional replication-associated protein binding sequence (“RABS”). 
     
     
         54 . The double-stranded DNA molecule of  claim 37 , wherein the double-stranded DNA molecule lacks a functional replication-associated protein (“RAP”) encoding sequence. 
     
     
         55 . The double-stranded DNA molecule of  claim 37 , wherein the double-stranded DNA molecule lacks a viral capsid protein encoding sequence. 
     
     
         56 . The double-stranded DNA molecule of  claim 37 , wherein the first inverted repeat or the second inverted repeat lacks at a functional RABS. 
     
     
         57 . (canceled) 
     
     
         58 . The double-stranded DNA molecule of  claim 37 , wherein the DNA sequence between the ITR closing base pair of the first inverted repeat and the ITR closing base pair of the second inverted repeat lacks a functional RABS. 
     
     
         59 . The double-stranded DNA molecule of  claim 37 , wherein the first inverted repeat lacks a functional RABS and the second inverted repeat lacks a functional RABS. 
     
     
         60 . The double-stranded DNA molecule of  claim 37 , wherein the double-stranded DNA molecule lacks a functional terminal resolution site (TRS). 
     
     
         61 . The double-stranded DNA molecule of  claim 37 , wherein the first inverted repeat or the second inverted repeat lack(s) a functional TRS. 
     
     
         62 . (canceled) 
     
     
         63 . The double-stranded DNA molecule of  claim 37 , wherein the DNA sequence between the ITR closing base pair of the first inverted repeat and the ITR closing base pair of the second inverted repeat lacks a functional TRS. 
     
     
         64 . The double-stranded DNA molecule of  claim 37 , wherein the first inverted repeat lacks a functional TRS and the second inverted repeat lacks a functional TRS. 
     
     
         65 . The double-stranded DNA molecule of  claim 53 , wherein the mobilization risk of the double-stranded DNA molecule when administered to a host is lower than control DNA molecules with the at least one functional RABS and/or with a functional TRS by 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50%. 
     
     
         66 . The double-stranded DNA molecule of  claim 60 , which lacks a functional TRS, wherein the mobilization risk of the double-stranded DNA molecule when administered to a host is lower than control DNA molecules with the TRS by 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50%. 
     
     
         67 . The double-stranded DNA molecule of  claim 53 , which lacks at least one functional RABS and lacks a functional TRS, wherein the mobilization risk of the double-stranded DNA molecule when administered to a host is lower than control DNA molecules with the at least one RABS and the TRS by 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50%. 
     
     
         68 . Isolated double-stranded DNA molecules of  claim 5 , wherein the isolated double-stranded DNA molecules are:
 a. free of fragments of the double-stranded DNA molecule; and/or   b. no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the isolated double-stranded DNA molecules; and/or   c. free of nucleic acid contaminants that are not fragments of the double-stranded DNA molecules; and/or   d. free of baculoviral DNA.   
     
     
         69 . (canceled) 
     
     
         70 . (canceled) 
     
     
         71 . Isolated double-stranded DNA molecule of  claim 68 , wherein:
 a. the nucleic acid contaminants that are not fragments of the double-stranded DNA molecules are less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the isolated double-stranded DNA molecules; and/or   b. the baculoviral DNA is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the isolated double-stranded DNA molecules.   
     
     
         72 . (canceled) 
     
     
         73 . (canceled) 
     
     
         74 . A delivery vehicle comprising the double-stranded DNA molecule of  claim 37 . 
     
     
         75 . The delivery vehicle of  claim 74 , wherein the delivery vehicle comprises a hybridosome, a liposome, or a lipid nanoparticle. 
     
     
         76 . A method for preparing a hairpin-ended DNA molecule, wherein the method comprises:
 a. culturing a host cell comprising the double-stranded DNA molecule of  claim 1  under conditions resulting in amplification of the double-stranded DNA molecule;   b. releasing the double-stranded DNA molecule from the host cell;   c. incubating the double-stranded DNA molecule with one or more nicking endonuclease recognizing the four restriction sites resulting in four nicks;   d. denaturing and thereby creating a DNA fragment that comprises the expression cassette and is flanked by the two single strand DNA overhangs;   e. annealing the single strand DNA overhangs intramolecularly and thereby creating a hairpinned inverted repeat on both ends of the DNA fragment resulting from step d.   
     
     
         77 . A method for preparing a hairpin-ended DNA, wherein the method comprises:
 a. culturing a host cell comprising the double-stranded DNA molecule of  claim 20  under conditions resulting in amplification of the double-stranded DNA molecule;   b. releasing the double-stranded DNA molecule from the host cell;   c. incubating the double-stranded DNA molecule with one or more nicking endonuclease recognizing the four restriction sites resulting in four nicks;   d. denaturing and thereby creating a DNA fragment that comprises the expression cassette and is flanked by the two single strand DNA overhangs;   e. annealing the single strand DNA overhangs intramolecularly and thereby creating a hairpinned inverted repeat on both ends of the DNA fragment resulting from step d;   f. incubating the double-stranded DNA molecule or the fragments resulting from step d with the restriction enzyme and thereby cleaving the double-stranded DNA molecule or a fragment of the double-stranded DNA molecule; and   g. incubating the fragments of the double-stranded DNA molecule with an exonuclease thereby digesting the fragments of the double-stranded DNA molecule except the fragment resulting from step e.   
     
     
         78 . A method for preparing a hairpin-ended DNA, wherein the method comprises:
 a. culturing a host cell comprising the double-stranded DNA molecule of  claim 24  under conditions resulting in amplification of the double-stranded DNA molecule;   b. releasing the double-stranded DNA molecule from the host cell;   c. incubating the double-stranded DNA molecule with one or more nicking endonuclease recognizing the first, second, third, and fourth restriction sites resulting in four nicks;   d. denaturing and thereby creating a DNA fragment that comprises the expression cassette and is flanked by the two single strand DNA overhangs;   e. annealing the single strand DNA overhangs intramolecularly and thereby creating a hairpinned inverted repeat on both ends of the DNA fragment resulting from step d;   f. incubating the double-stranded DNA molecule or the fragments resulting from step d with one or more nicking endonuclease recognizing the fifth and sixth restriction sites resulting in the break in the double stranded DNA molecule; and   g. incubating the fragments of the double-stranded DNA molecule with an exonuclease thereby digesting the fragments of the double-stranded DNA molecule except the fragment resulting from step e.   
     
     
         79 . The method of  claim 76 , further comprising: h. repairing the nicks with a ligase to form a circular DNA. 
     
     
         80 . The method of  claim 76 , wherein:
 a. the steps are performed in the order in which they appear in the  claim ; and/or   b. the hairpin-ended DNA consists of two hairpin ends; and/or   c. the hairpin-ended DNA is a viral genome.   
     
     
         81 . (canceled) 
     
     
         82 . (canceled) 
     
     
         83 . The method of  claim 80 , wherein:
 a. the viral genome is parvovirus genome; and/or   b. the parvovirus is a Dependoparvovirus, a Bocaparvovirus, an Erythroparvovirus, a Protoparvovirus, or a Tetraparvovirus.   
     
     
         84 . (canceled) 
     
     
         85 . The method of  claim 76 , wherein the DNA molecule:
 a. lacks any functional RABS; and/or   b. lacks any functional TRS; and/or   c. lacks or has reduced (by at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or at least 99.9%) P5 AAV promoter activity; and/or   d. lacks any other elements required for viral replication.   
     
     
         86 . The method of  claim 85 , wherein the DNA molecule is characterized in that:
 a. any RABS in the DNA molecule is deleted or mutated to be functionally inactive; and/or   b. any TRS in the DNA molecule is deleted or mutated to be functionally inactive; and/or   c. any P5 AAV promoter is deleted or mutated to be functionally inactive; and/or   d. any other element required for viral replication in the DNA molecule is deleted or mutated to be functionally inactive.   
     
     
         87 . The method of  claim 86 , wherein the DNA molecule is characterized in that the deletion is only of the RABS and/or TRS and/or any P5 AAV promoter and/or any other element required for replication but does not delete an entire stem/loop structure. 
     
     
         88 . (canceled) 
     
     
         89 . (canceled) 
     
     
         90 . (canceled) 
     
     
         91 . The method of  claim 76 , wherein the DNA molecule is characterized in that the 5′ end of the top strand of the expression cassette is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100 nucleotides, greater than 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 300 nucleotides, or at least 400 nucleotides apart from the 3′ top strand end of the inverted repeat. 
     
     
         92 . The method of  claim 76 , wherein the DNA molecule is characterized in that the 3′ end of the top strand of the expression cassette is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100 nucleotides, greater than 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 300 nucleotides, or at least 400 nucleotides apart from the 5′ top strand end of the inverted repeat. 
     
     
         93 . (canceled) 
     
     
         94 . The method of  claim 76 , wherein the DNA molecule is characterized in that the bottom strand is in anti-sense orientation with respect to an open reading frame in the expression cassette. 
     
     
         95 . The method of  claim 76 , wherein the DNA molecule is characterized in that the the top strand is in anti-sense orientation with respect to an open reading frame in the expression cassette. 
     
     
         96 . (canceled) 
     
     
         97 . A DNA molecule resulting from the method of  claim 76 . 
     
     
         98 . The method of  claim 76 , wherein the DNA molecule:
 a. comprises a first inverted repeat that lacks any transcriptional activity; and/or   b. comprises a second inverted repeat that lacks any transcriptional activity; and/or   c. lacks or has reduced (by at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or at least 99.9%) P5 AAV promoter activity.   
     
     
         99 . The method of  claim 76 , wherein the DNA molecule:
 a. comprises a first inverted repeat that lacks transcriptional activity; and/or   b. comprises a second inverted repeat that lacks transcriptional activity; and/or   c. lacks any other elements in the inverted repeats that are transcriptionally active.   
     
     
         100 . The method of  claim 76 , wherein the DNA molecule is characterized in that:
 a. any CpG motif in the first inverted repeat is deleted or mutated to be transcriptionally inactive; and/or   b. any CpG motif in the second inverted repeat is deleted or mutated to be transcriptionally inactive; and/or   c. any other element required for transcriptional activity in the inverted repeat is deleted or mutated to be functionally inactive.   
     
     
         101 . The method of  claim 76 , wherein the DNA molecule is characterized in that:
 a. any CpG motif in the first inverted repeat is deleted or mutated to be reduce toll like receptor 9 binding; and/or   b. any CpG motif in the second inverted repeat is deleted or mutated to reduce toll like receptor 9 binding; and/or   c. any other element required for toll like receptor 9 binding in the inverted repeat is deleted or mutated to be functionally inactive.