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US 2024/0229071 A1Published application
Expression vectors, bacterial sequence-free vectors, and methods of making and using the same
AssigneeMediphage Bioceuticals, Inc.
InventorsRoderick SLAVCEV, Nafiseh NAFISSI
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Abstract
The present disclosure provides expression vectors, bacterial sequence-free vectors, such as ministring DNA (msDNA), and methods of making the bacterial sequence-free vectors, including with vector production systems. The present disclosure also provides compositions comprising the vectors, and uses of the vectors and compositions.
Claims34
1-135. (canceled)
An expression vector comprising:
- (a) a backbone sequence,
- (b) a sequence comprising:
- (i) an expression cassette comprising a nucleic acid sequence of interest,
- (ii) a first target sequence for a first recombinase flanking the 5′ side of the expression cassette,
- (iii) a second target sequence for the first recombinase flanking the 3′ side of the expression cassette, and
- (iv) one or more additional target sequences for one or more additional recombinases integrated within the first and second target sequences in non-binding regions for the first recombinase, and
- (c) one or more of:
- (i) an endonuclease target sequence integrated within the first and/or second target sequences for the first recombinase in non-binding regions for the first recombinase and the one or more additional recombinases, wherein the endonuclease target sequence is between the backbone sequence and cleavage sites for the first recombinase and the one or more additional recombinases,
- (ii) a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:
- 12 integrated between the 3′ end of the first target sequence for the first recombinase and the 5′ end of another enhancer or a promoter in the expression cassette,
- (iii) a cytomegalovirus (CMV) enhancer integrated between the 3′ end of the first target sequence for the first recombinase and the 5′ end of a promoter in the expression cassette,
- (iv) a 5′ untranslated region (5′UTR) comprising an intron, wherein the 5′UTR is integrated in the expression cassette between a promoter and the nucleic acid sequence of interest,
- (v) a vertebrate chromatin insulator integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal,
- (vi) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal,
- (vii) a scaffold/matrix attachment region (S/MAR) integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, or
- (viii) a DNA nuclear targeting sequence (DTS) integrated within the first and/or second target sequences for the first recombinase in non-binding regions for the first recombinase and the one or more additional recombinases, wherein the DTS is between the expression cassette and cleavage sites for the first recombinase and the one or more additional recombinases.
13 more elements
A bacterial sequence-free vector comprising:
- (a) an expression cassette comprising a nucleic acid sequence of interest, and
- (b) one or more of:
- (i) a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:
- 12 located 5′ to another enhancer or a promoter in the expression cassette,
- (ii) a CMV enhancer located 5′ to a promoter in the expression cassette,
- (iii) a 5′UTR comprising an intron, wherein the 5′UTR is integrated in the expression cassette between a promoter and the nucleic acid sequence of interest,
- (iv) a vertebrate chromatin insulator integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal,
- (v) a WPRE integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal,
- (vi) a S/MAR integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, or
- (vii) a DTS located 5′ to the expression cassette.
7 more elements
A polynucleotide comprising a nucleic acid sequence at least about 90% identical to any one of SEQ ID NOs:
- 1, 2, 3, 5, 12-18, 35-39, and 46.
An expression vector comprising:
- a polynucleotide comprising a nucleic acid sequence at least about 90% identical to any one of SEQ ID NOs:
- 2, 3, and 5, and a polynucleotide comprising a nucleic acid sequence at least about 90% identical to any one of SEQ ID NOs:
- 13-18.
Dependencies are inferred from the claim text.