Nannochloropsis-producing viruses and methods and compositions for making the same
AssigneeInsmed Incorporated
InventorsAnthony BERNDT, Allan KASPAR, Brian KASPAR, Samit VARMA, Gretchen THOMSEN
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Abstract
The present disclosure relates in part to Nannochloropsis microalgae that produce recombinant viruses. Expression cassettes, transgenes and methods for producing recombinant viruses in the Nannochloropsis are also provided. In particular, methods and materials are provided for producing recombinant adeno associated viruses (AAV) in Nannochloropsis. In some embodiments described herein, the Nannochloropsis is Nannochloropsis oceanica.
Claims231
An expression cassette, comprising,
- (i) a first Nannochloropsis-endogenous promoter comprising a first end and a second end, (ii) a first gene coding sequence operably linked to the first end of the first Nannochloropsis-endogenous promoter, (iii) a first Nannochloropsis-endogenous 3′ untranslated region (UTR) downstream of the first gene coding sequence and operably linked thereto, (iv) an antibiotic resistance gene coding sequence,
- wherein the antibiotic resistance gene coding sequence is operably linked to the first Nannochloropsis-endogenous promoter or a second Nannochloropsis-endogenous promoter, and
- wherein the first gene coding sequence is an adeno-associated virus (AAV) cap coding sequence.
The expression cassette of any one of claims 71-93, wherein the second 3′ UTR is the VCP1 3′ UTR, the VCP2 3′ UTR, the LDSP 3′ UTR, the heat shock protein 3′ UTR or the cellulose synthase (CS) 3′ UTR.
An expression cassette, comprising,
- (i) a constitutively active Nannochloropsis-endogenous bidirectional promoter comprising a first end and a second end,
- (ii) a first gene coding sequence operably linked to the first end of the constitutively active Nannochloropsis-endogenous bidirectional promoter, wherein the first gene coding sequence is a Rep52 coding sequence,
- (iii) a first Nannochloropsis-endogenous 3′ untranslated region (UTR) downstream of the first gene coding sequence and operably linked thereto,
- (iv) a second gene coding sequence operably linked to the second end of the constitutively active Nannochloropsis-endogenous bidirectional promoter, wherein the second gene coding sequence is selected from an AAV assembly activating protein (AAP) gene coding sequence, and an antibiotic resistance gene coding sequence,
- (v) a second Nannochloropsis-endogenous 3′ untranslated region (UTR) downstream of the second gene coding sequence and operably linked thereto,
- (vi) an inducible Nannochloropsis-endogenous bidirectional promoter comprising a first end and a second end,
- (vii) a third gene coding sequence operably linked to the first end of the inducible Nannochloropsis-endogenous bidirectional promoter, wherein the third gene coding sequence is a Rep78 coding sequence,
- (viii) a third Nannochloropsis-endogenous 3′ untranslated region (UTR) downstream of the third coding sequence and operably linked thereto,
- (ix) a fourth gene coding sequence operably linked to the second end of the inducible Nannochloropsis-endogenous bidirectional promoter, wherein the fourth gene coding sequence is selected from an adenoviral gene coding sequence and an antibiotic resistance gene coding sequence, and
- (x) a fourth Nannochloropsis-endogenous 3′ untranslated region (UTR) downstream of the fourth coding sequence and operably linked thereto,
- wherein the expression cassette comprises a single antibiotic resistance gene coding sequence.
A Nannochloropsis microalgae that produces a recombinant adeno-associated virus (rAAV).
Dependencies are inferred from the claim text.