Closed-ended DNA vectors obtainable from cell-free synthesis and process for obtaining ceDNA vectors
The application describes methods for synthetic synthesis and cell-free synthesis of DNA vectors, particularly closed-ended DNA vectors (e.g., ceDNA vectors) having linear and continuous structure for delivery and expression of a transgene. The present invention relates to an in vitro process for production of closed-ended DNA vectors, corresponding DNA vector products produced by the methods and uses thereof, and oligonucleotides and kits useful in the process of the invention. DNA vectors produced using the methods described herein are free from unwanted side effects due to contaminants introduced during production in cell lines, for example, bacterial or insect cell lines. Further provided herein are methods and cell lines for reliable gene expression in vitro, ex vivo and in vivo using the ceDNA vectors synthesized using the methods herein.
1. A cell-free method of preparing a non-viral, linear and closed-ended DNA (ceDNA) vector comprising:
providing a first single-stranded inverted terminal repeat (ITR) molecule comprising a first ITR;
providing a second single-stranded ITR molecule comprising a second ITR, wherein the first ITR and the second ITR are synthetic;
providing a double-stranded polynucleotide comprising a double-stranded expression cassette sequence, wherein the double-stranded polynucleotide comprising the double-stranded expression cassette is obtained by excision from a double-stranded DNA construct; and
ligating the 5′ and 3′ ends of the first ITR molecule to a first end of the double-stranded polynucleotide and ligating the 5′ and 3′ ends of the second ITR molecule to a second end of the double-stranded polynucleotide to form the ceDNA vector, wherein:
the first ITR molecule comprises a first overhang region that is complementary to an overhang region on the first end of the double-stranded polynucleotide, and the second ITR molecule comprises a second overhang region that is complementary to an overhang region on the second end of the double-stranded polynucleotide;
each of the first overhang region and the second overhang region is located in an A-stem of the respective ITR; and
ITR D regions are present in the double-stranded expression cassette sequence;
thereby preparing the ceDNA vector without using cells.
2. The method of claim 1 , further comprising a step of synthesizing at least one of the first ITR and the second ITR using in vitro DNA synthesis prior to the providing steps.
3. The method of claim 1 , wherein, within the double-stranded DNA construct, the double-stranded polynucleotide comprising the double-stranded expression cassette sequence is flanked at the 5′ end by a first restriction endonuclease cleavage site and at the 3′ end by a second restriction endonuclease cleavage site.
4. The method of claim 1 , wherein the double-stranded DNA construct is selected from the group consisting of: a bacmid, a plasmid, a minicircle, and a linear double-stranded DNA molecule.
5. The method of claim 1 , wherein at least one of the first ITR and the second ITR are annealed prior to ligation to the double-stranded polynucleotide comprising the expression cassette sequence.
6. The method of claim 1 , wherein the ligation is effected by T4 ligase or an AAV Rep protein.
7. The method of claim 1 , wherein the first ITR is selected from the group consisting of a wild-type ITR and a modified ITR, and/or the second ITR is selected from the group consisting of a wild-type ITR and a modified ITR.
8. The method of claim 1 , wherein at least one of the first ITR and the second ITR comprises at least one RBE site.
9. The method of claim 1 , wherein at least one of the first ITR and the second ITR is an AAV ITR or an AAV-derived ITR.
10. The method of claim 1 , wherein the double-stranded expression cassette sequence comprises at least one cis-regulatory element.
11. The method of claim 1 , wherein the double-stranded expression cassette sequence comprises a transgene sequence.
12. The method of claim 11 , wherein the transgene sequence encodes a protein.
13. The method of claim 1 , wherein the double stranded polynucleotide comprises at least one RBE element and an ITR D region.
14. The method of claim 1 , wherein the method steps are all performed in a manner that does not involve replication of the ceDNA vector by or inside of a cell or using a cellular extract.
15. The method of claim 1 , wherein the double-stranded polynucleotide comprises a first Rep binding element (RBE) and a second RBE.
16. The method of claim 1 , wherein each of the first overhang region and the second overhang region is located in the A-stem of the respective ITR between: hairpin loop sequences B and C, and a Rep binding element (RBE).