METHODS AND COMPOSITIONS FOR GENOMIC INTEGRATION

1 . A method of integrating a nucleic acid sequence into a genome of a cell, the method comprising introducing a recombinant mRNA or a vector encoding an mRNA into the cell, wherein the mRNA comprises:

(a) an insert sequence, wherein the insert sequence comprises

(i) an exogenous sequence, or

(ii) a sequence that is a reverse complement of the exogenous sequence;

(b) a 5′ UTR sequence and a 3′ UTR sequence downstream of the 5′ UTR sequence; wherein the 5′ UTR sequence or the 3′ UTR sequence comprises a binding site for a human ORF protein, and

wherein the insert sequence is integrated into the genome of the cell.

2 . The method of claim 1 , wherein the 5′ UTR sequence or the 3′ UTR sequence comprises a binding site for human ORF2p.

3 . A method for integrating a nucleic acid sequence into the genome of an immune cell, the method comprising introducing a recombinant mRNA or a vector encoding an mRNA, wherein the mRNA comprises:

(a) an insert sequence, wherein the insert sequence comprises (i) an exogenous sequence or (ii) a sequence that is a reverse complement of the exogenous sequence;

(b) 5′ UTR sequence and a 3′ UTR sequence downstream of the 5′ UTR sequence, wherein the 5′ UTR sequence or the 3′ UTR sequence comprises an endonuclease binding site and/or a reverse transcriptase binding site, and

wherein the transgene sequence is integrated into the genome of the immune cell.

4 . A method for integrating a nucleic acid sequence into the genome of a cell, the method comprising introducing a recombinant mRNA or a vector encoding an mRNA, wherein the mRNA comprises:

(a) an insert sequence, wherein the insert sequence comprises (i) an exogenous sequence or (ii) a sequence that is a reverse complement of the exogenous sequence;

(b) a 5′ UTR sequence, a sequence of a human retrotransposon downstream of the 5′ UTR sequence, and a 3′ UTR sequence downstream of the sequence of a human retrotransposon;

wherein the 5′ UTR sequence or the 3′ UTR sequence comprises an endonuclease binding site and/or a reverse transcriptase binding site, and

wherein the sequence of a human retrotransposon encodes for two proteins that are translated from a single RNA containing two ORFs, and

wherein the insert sequence is integrated into the genome of the cell.

5 . The method of claim 3 or 4 , wherein the 5′ UTR sequence or the 3′ UTR sequence comprises an ORF2p binding site.

6 . The method of claim 2 or 5 , wherein the ORF2p binding site is a poly A sequence in the 3′ UTR sequence.

7 . The method of any one of claims 1 - 3 , wherein the mRNA comprises a sequence of a human retrotransposon.

8 . The method of claim 7 , wherein the sequence of a human retrotransposon is downstream of the 5′ UTR sequence.

9 . The method of claim 7 or 8 , wherein the sequence of a human retrotransposon is upstream of the 3′ UTR sequence.

10 . The method of any one of claims 7 - 9 , wherein the sequence of a human retrotransposon encodes for two proteins that are translated from a single RNA containing two ORFs.

11 . The method of claim 4 or 10 , wherein the two ORFs are non-overlapping ORFs.

12 . The method of claim 4 , 10 or 11 , wherein the two ORFs are ORF1 and ORF2.

13 . The method of claim 12 , wherein the ORF1 encodes ORF1p and ORF2 encodes ORF2p.

14 . The method of any one of claims 4 - 13 , wherein the sequence of a human retrotransposon comprises a sequence of a non-LTR retrotransposon.

15 . The method of any one of claims 4 - 13 , wherein the sequence of a human retrotransposon encodes comprises a LINE-1 retrotransposon.

16 . The method of claim 15 , wherein the LINE-1 retrotransposon is a human LINE-1 retrotransposon.

17 . The method of any one of claims 4 - 16 , wherein the sequence of a human retrotransposon comprises a sequence encoding an endonuclease and/or a reverse transcriptase.

18 . The method of claim 17 , wherein the endonuclease and/or a reverse transcriptase is ORF2p.

19 . The method of claim 17 , wherein the reverse transcriptase is a group II intron reverse transcriptase domain.

20 . The method of claim 17 , wherein the endonuclease and/or a reverse transcriptase is a minke whale endonuclease and/or a reverse transcriptase.

21 . The method of any one of claim 4 - 16 or 20 , wherein the sequence of a human retrotransposon comprises a sequence encoding ORF2p.

22 . The method of claim 21 , wherein the insert sequence is integrated into the genome at a poly T site using specificity of an endonuclease domain of the ORF2p.

23 . The method of claim 22 , wherein the poly T site comprises the sequence TTTTTA.

24 . The method of any one of claims 4 - 23 , wherein (i) the sequence of a human retrotransposon comprises a sequence encoding ORF1p, (ii) the mRNA does not comprise a sequence encoding ORF1p, or (iii) the mRNA comprises a replacement of the sequence encoding ORF1p with a 5′ UTR sequence from the complement gene.

25 . The method of any one of claims 1 - 24 , wherein the mRNA comprises a first mRNA molecule encoding ORF1p, and a second mRNA molecule encoding an endonuclease and/or a reverse transcriptase.

26 . The method of any one of claims 1 - 24 , wherein the mRNA is an mRNA molecule comprising a first sequence encoding ORF1p, and a second sequence encoding an endonuclease and/or a reverse transcriptase.

27 . The method of claim 26 , wherein the first sequence encoding ORF1p and the second sequence encoding an endonuclease and/or a reverse transcriptase are separated by a linker sequence.

28 . The method of claim 27 , wherein the linker sequence comprises an internal ribosome entry sequence (IRES).

29 . The method of claim 28 , wherein the IRES is an IRES from CVB3 or EV71.

30 . The method of claim 27 , wherein the linker sequence encodes a self-cleaving peptide sequence.

31 . The method of claim 27 , wherein the linker sequence encodes a T2A, a E2A or a P2A sequence

32 . The method of any one of claims 1 - 31 , wherein the sequence of a human retrotransposon comprises a sequence that encodes ORF1p fused to an additional protein sequence and/or a sequence that encodes ORF2p fused to an additional protein sequence.

33 . The method of claim 32 , wherein the ORF1p and/or the ORF2p is fused to a nuclear retention sequence.

34 . The method of claim 33 , wherein the nuclear retention sequence is an Alu sequence.

35 . The method of claim 32 , wherein the ORF1p and/or the ORF2p is fused to an MS2 coat protein.

36 . The method of any one of claims 1 - 35 , wherein the 5′ UTR sequence or the 3′ UTR sequence comprises at least one, two, three or more MS2 hairpin sequences.

37 . The method of any one of claims 17 - 36 , wherein the 5′ UTR sequence or the 3′ UTR sequence comprises a sequence that promotes or enhances interaction of a poly A tail of the mRNA with the endonuclease and/or a reverse transcriptase.

38 . The method of any one of claims 17 - 37 , wherein the 5′ UTR sequence or the 3′ UTR sequence comprises a sequence that promotes or enhances interaction of a poly-A-binding protein (PABP) with the endonuclease and/or a reverse transcriptase.

39 . The method of any one of claims 17 - 38 , wherein the 5′ UTR sequence or the 3′ UTR sequence comprises a sequence that increases specificity of the endonuclease and/or a reverse transcriptase to the mRNA relative to another mRNA expressed by the cell.

40 . The method of any one of claims 1 - 32 , wherein the 5′ UTR sequence or the 3′ UTR sequence comprises an Alu element sequence.

41 . The method of any one of claims 26 - 40 , wherein the first sequence encoding ORF1p and the second sequence encoding an endonuclease and/or a reverse transcriptase have the same promoter.

42 . The method of any one of claims 24 - 41 , wherein the insert sequence has a promoter that is different from the promoter of the first sequence encoding ORF1p.

43 . The method of any one of claims 17 - 42 , wherein the insert sequence has a promoter that is different from the promoter of the second sequence encoding an endonuclease and/or a reverse transcriptase.

44 . The method of any one of claims 26 - 43 , wherein the first sequence encoding ORF1p and/or the second sequence encoding an endonuclease and/or a reverse transcriptase have a promoter or transcription initiation site selected from the group consisting of an inducible promoter, a CMV promoter or transcription initiation site, a T7 promoter or transcription initiation site, an EF1a promoter or transcription initiation site and combinations thereof.

45 . The method of any one of claims 1 - 44 , wherein the insert sequence has a promoter or transcription initiation site selected from the group consisting of an inducible promoter, a CMV promoter or transcription initiation site, a T7 promoter or transcription initiation site, an EF1a promoter or transcription initiation site and combinations thereof.

46 . The method of any one of claims 26 - 45 , wherein the first sequence encoding ORF1p and the second sequence encoding an endonuclease and/or a reverse transcriptase are codon optimized for expression in a human cell.

47 . The method of any one of claims 1 - 46 , wherein the mRNA comprises a WPRE element.

48 . The method of any one of claims 1 - 47 , wherein the mRNA comprises a selection marker.

49 . The method of any one of claims 1 - 48 , wherein the mRNA comprises a sequence encoding an affinity tag.

50 . The method of claim 49 , wherein the affinity tag is linked to the sequence encoding an endonuclease and/or a reverse transcriptase.

51 . The method of any one of claims 1 - 50 , wherein the 3′ UTR comprises a poly A sequence or wherein a poly A sequence is added to the mRNA in vitro.

52 . The method of claim 51 , wherein the poly A sequence is downstream of a sequence encoding an endonuclease and/or a reverse transcriptase.

53 . The method of claim 51 or 52 , wherein the insert sequence is upstream of the poly A sequence.

54 . The method of any one of claims 1 - 53 , wherein the 3′ UTR sequence comprises the insert sequence.

55 . The method of any one of claims 1 - 54 , wherein the insert sequence comprises a sequence that is a reverse complement of the sequence encoding the exogenous polypeptide.

56 . The method of any one of claims 1 - 55 , wherein the insert sequence comprises a polyadenylation site.

57 . The method of any one of claims 1 - 56 , wherein the insert sequence comprises an SV40 polyadenylation site.

58 . The method of any one of claims 1 - 57 , wherein the insert sequence comprises a polyadenylation site upstream of the sequence that is a reverse complement of the sequence encoding the exogenous polypeptide.

59 . The method of any one of claims 1 - 58 , wherein the insert sequence is integrated into the genome at a locus that is not a ribosomal locus.

60 . The method of any one of claims 1 - 58 , wherein the insert sequence integrates into a gene or regulatory region of a gene, thereby disrupting the gene or downregulating expression of the gene.

61 . The method of any one of claims 1 - 58 , wherein the insert sequence integrates into a gene or regulatory region of a gene, thereby upregulating expression of the gene.

62 . The method of any one of claims 1 - 58 , wherein the insert sequence integrates into the genome and replaces a gene.

63 . The method of any one of claims 1 - 62 , wherein the insert sequence is stably integrated into the genome.

64 . The method of any one of claims 1 - 63 , wherein the insert sequence is retrotransposed into the genome.

65 . The method of any one of claims 1 - 64 , wherein the insert sequence is integrated into the genome by cleavage of a DNA strand of a target site by an endonuclease encoded by the mRNA.

66 . The method of any one of claims 1 - 65 , wherein the insert sequence is integrated into the genome via target-primed reverse transcription (TPRT).

67 . The method of any one of claims 1 - 65 , wherein the insert sequence is integrated into the genome via reverse splicing of the mRNA into a DNA target site of the genome.

68 . The method of any one of claim 1 or 4 - 67 , wherein the cell is an immune cell.

69 . The method of claim 3 or 68 , wherein the immune cell is a T cell or a B cell.

70 . The method of claim 3 or 68 , wherein the immune cell is a myeloid cell.

71 . The method of claim 3 or 68 , wherein the immune cell is selected from a group consisting of a monocyte, a macrophage, a dendritic cell, a dendritic precursor cell, and a macrophage precursor cell.

72 . The method of any one of claims 1 - 71 , wherein the mRNA is a self-integrating mRNA.

73 . The method of any one of claims 1 - 72 , wherein the method comprises introducing into the cell the mRNA.

74 . The method of any one of claims 1 - 72 , wherein the method comprises introducing into the cell the vector encoding the mRNA.

75 . The method of any one of claims 1 - 74 , wherein the method comprises introducing the mRNA or the vector encoding the mRNA into a cell ex vivo.

76 . The method of claim 75 , wherein the method further comprises administering the cell to a human subject.

77 . The method of any one of claims 1 - 74 , wherein the method comprises administering the mRNA or the vector encoding the mRNA to a human subject

78 . The method of claim 76 or 77 , wherein an immune response is not elicited in the human subject.

79 . The method of claim 76 or 77 , wherein the mRNA or the vector is substantially non-immunogenic.

80 . The method of any one of claims 1 - 79 , wherein the vector is a plasmid or a viral vector.

81 . The method of any one of claims 1 - 79 , wherein the vector comprises a non-LTR retrotransposon.

82 . The method of any one of claims 1 - 79 , wherein the vector comprises a human L1 element.

83 . The method of any one of claims 1 - 79 , wherein the vector comprises a L1 retrotransposon ORF1 gene.

84 . The method of any one of claims 1 - 79 , wherein the vector comprises a L1 retrotransposon ORF2 gene.

85 . The method of any one of claims 1 - 79 , wherein the vector comprises a L1 retrotransposon.

86 . The method of any one of claims 1 - 85 , wherein the mRNA is at least about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 kilobases.

87 . The method of any one of claims 1 - 86 , wherein the mRNA is a most about 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5 kilobases.

88 . The method of any one of claims 1 - 87 , wherein the mRNA comprises a sequence that inhibits or prevents degradation of the mRNA.

89 . The method of claim 88 , wherein the sequence that inhibits or prevents degradation of the mRNA inhibits or prevents degradation of the mRNA by an exonuclease or an RNAse.

90 . The method of claim 88 , wherein the sequence that inhibits or prevents degradation of the mRNA is a G quadruplex, pseudoknot or triplex sequence.

91 . The method of claim 88 , wherein the sequence the sequence that inhibits or prevents degradation of the mRNA is an exoribonuclease-resistant RNA structure from a flaviviral RNA or an ENE element from KSV.

92 . The method of claim 88 , wherein the sequence that inhibits or prevents degradation of the mRNA inhibits or prevents degradation of the mRNA by a deadenylase.

93 . The method of claim 88 , wherein the sequence that inhibits or prevents degradation of the mRNA comprises non-adenosine nucleotides within or at a terminus of a poly A tail of the mRNA.

94 . The method of claim 88 , wherein the sequence that inhibits or prevents degradation of the mRNA increases stability of the mRNA.

95 . The method of any one of claims 1 - 94 , wherein the exogenous sequence comprises a sequence encoding an exogenous polypeptide.

96 . The method of claim 95 , wherein the sequence encoding an exogenous polypeptide is not in frame with a sequence encoding an endonuclease and/or a reverse transcriptase

97 . The method of claim 95 or 96 , wherein the sequence encoding an exogenous polypeptide is not in frame with a sequence encoding an endonuclease and/or a reverse transcriptase.

98 . The method of any one of claims 95 - 97 , wherein the exogenous sequence does not comprise introns.

99 . The method of any one of claims 95 - 98 , wherein the exogenous sequence comprises a sequence encoding an exogenous polypeptide selected from the group consisting of an enzyme, a receptor, a transport protein, a structural protein, a hormone, an antibody, a contractile protein and a storage protein.

100 . The method of any one of claims 95 - 98 , wherein the exogenous sequence comprises a sequence encoding an exogenous polypeptide selected from the group consisting of a chimeric antigen receptor (CAR), a ligand, an antibody, a receptor, and an enzyme.

101 . The method of any one of claims 1 - 94 , wherein the exogenous sequence comprises a regulatory sequence.

102 . The method of claim 101 , wherein the regulatory sequence comprises a cis-acting regulatory sequence.

103 . The method of claim 101 , wherein the regulatory sequence comprises a cis-acting regulatory sequence selected from the group consisting of an enhancer, a silencer, a promoter or a response element.

104 . The method of claim 101 , wherein the regulatory sequence comprises a trans-acting regulatory sequence.

105 . The method of claim 101 , wherein the regulatory sequence comprises a trans-acting regulatory sequence that encodes a transcription factor.

106 . The method of any one of the claims 1 - 105 , wherein integration of the insert sequence does not adversely affect cell health.

107 . The method of any one of the claims 1 - 106 , wherein the endonuclease, the reverse transcriptase or both are capable of site specific integration of the insert sequence.

108 . The method of any one of the claims 1 - 107 , wherein the mRNA comprises a sequence encoding an additional nuclease domain or a nuclease domain that is not derived from ORF2.

109 . The method of any one of the claims 1 - 107 , wherein the mRNA comprises a sequence encoding a megaTAL nuclease domain, a TALEN domain, a Cas9 domain, a zinc finger binding domain from an R2 retroelement, or a DNA binding domain that binds to repetitive sequences such as a Rep78 from AAV.

110 . The method of any one of claims 17 - 109 , wherein the endonuclease comprises a mutation that reduces activity of the endonuclease compared to the endonuclease without the mutation.

111 . The method of claim 110 , wherein the endonuclease is an ORF2p endonuclease and the mutation is S228P.

112 . The method of any one of the claims 17 - 111 , wherein the mRNA comprises a sequence encoding a domain that increases fidelity and/or processivity of the reverse transcriptase.

113 . The method of any one of the claims 17 - 111 , wherein the reverse transcriptase is a reverse transcriptase from a retroelement other than ORF2 or reverse transcriptase that has higher fidelity and/or processivity compared to a reverse transcriptase of ORF2p.

114 . The method of claim 113 , wherein the reverse transcriptase is a group II intron reverse transcriptase.

115 . The method of claim 114 , wherein the group II intron reverse transcriptase is a group IIA intron reverse transcriptase, a group IIB intron reverse transcriptase, or a group IIC intron reverse transcriptase.

116 . The method of claim 114 , wherein the group II intron reverse transcriptase is TGIRT-II or TGIRT-III.

117 . The method of any one of the claims 1 - 116 , wherein the mRNA comprises a sequence comprising an Alu element and/or a ribosome binding aptamer.

118 . The method of any one of the claims 1 - 117 , wherein the mRNA comprises a sequence encoding a polypeptide comprising a DNA binding domain

119 . The method of any one of the claims 1 - 118 , wherein the 3′ UTR sequence is derived from a viral 3′ UTR or a beta-globin 3′ UTR.

120 . A composition comprising a recombinant mRNA or vector encoding an mRNA, wherein the mRNA comprises a human LINE-1 transposon sequence comprising

(i) a human LINE-1 transposon 5′ UTR sequence,

(ii) a sequence encoding ORF1p downstream of the human LINE-1 transposon 5′ UTR sequence,

(iii) an inter-ORF linker sequence downstream of the sequence encoding ORF1p,

(iv) a sequence encoding ORF2p downstream of the inter-ORF linker sequence, and

(v) a 3′ UTR sequence derived from a human LINE-1 transposon downstream of the sequence encoding ORF2p;

wherein the 3′ UTR sequence comprises an insert sequence, wherein the insert sequence is a reverse complement of a sequence encoding an exogenous polypeptide or a reverse complement of a sequence encoding an exogenous regulatory element.

121 . The composition of claim 120 , wherein the insert sequence integrates into the genome of a cell when introduced into the cell.

122 . The composition of claim 121 , wherein the insert sequence integrates into a gene associated a condition or disease, thereby disrupting the gene or downregulating expression of the gene.

123 . The composition of claim 121 , wherein the insert sequence integrates into a gene, thereby upregulating expression of the gene.

124 . The composition of claim 121 , wherein the mRNA comprises a sequence with at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 35-50.

125 . The composition of claim 121 , wherein the recombinant mRNA or vector encoding the mRNA is isolated or purified.

126 . A composition comprising a nucleic acid comprising a nucleotide sequence encoding (a) a long interspersed nuclear element (LINE) polypeptide, wherein the LINE polypeptide includes human ORF1p and human ORF2p; and (b) an insert sequence, wherein the insert sequence is a reverse complement of a sequence encoding an exogenous polypeptide or a reverse complement of a sequence encoding an exogenous regulatory element, wherein the composition is substantially non-immunogenic.

127 . The composition of claim 126 , wherein the composition comprises human ORF1p and human ORF2p proteins.

128 . The composition of claim 126 or 127 , wherein the composition comprises a ribonucleoprotein (RNP) comprising human ORF1p and human ORF2p complexed to the nucleic acid.

129 . The composition of any one of claims 127 - 128 , wherein the nucleic acid is mRNA.

130 . A composition comprising a cell comprising the composition of any one of the claims 120 - 129 .

131 . The composition of claim 130 , wherein the cell is an immune cell.

132 . The composition of claim 131 , wherein the immune cell is a T cell or a B cell.

133 . The composition of claim 131 , wherein the immune cell is a myeloid cell.

134 . The composition of claim 131 , wherein the immune cell is selected from a group consisting of a monocyte, a macrophage, a dendritic cell, a dendritic precursor cell, and a macrophage precursor cell.

135 . The composition of any one of claims 120 - 134 , wherein the insert sequence is a reverse complement of a sequence encoding an exogenous polypeptide and the exogenous polypeptide is a chimeric antigen receptor (CAR).

136 . A pharmaceutical composition comprising the composition of any one of the claims 120 - 135 , and a pharmaceutically acceptable excipient.

137 . The pharmaceutical composition of claim 136 for use in gene therapy.

138 . The pharmaceutical composition of claim 136 for use in the manufacture of a medicament for treating a disease or condition.

139 . The pharmaceutical composition of claim 136 for use in treating a disease or condition.

140 . A method of treating a disease in a subject, comprising administering the pharmaceutical composition of claim 136 to a subject with a disease or condition.

141 . The method of claim 140 , wherein the method increases an amount or activity of a protein or functional RNA in the subject.

142 . The method of claim 140 or 141 , wherein the subject has a deficient amount or activity of a protein or functional RNA.

143 . The method of claim 142 , wherein the deficient amount or activity of a protein or functional RNA is associated with or causes the disease or condition.

144 . The method of any one of claims 140 - 143 , further comprising administering an agent that inhibits human silencing hub (HUSH) complex, an agent that inhibits FAM208A, or an agent that inhibits TRIM28.

145 . The method of claim 144 , wherein the agent that inhibits human silencing hub (HUSH) complex is an agent that inhibits Periphilin, TASOR and/or MPP8.

146 . The method of claim 144 , wherein the agent that inhibits human silencing hub (HUSH) complex inhibits assembly of the HUSH complex.

147 . The method of any one of claims 140 - 146 , further comprising administering an agent that inhibits fanconia anemia complex.