EP-4741499-A1 - PROTECTED DNA AND METHODS FOR THE PRODUCTION THEREOF

Abstract

Protected DNA comprising a single-stranded DNA (ssDNA) cassette is provided. Further provided are uses of the protected DNA, methods for producing protected DNA, products generated in performing such methods (including intermediate and final products), and kits for use in such methods.

Inventors

• BOUCHAREB, Amine
• RISTIN, Anca-Paula
• WALKER, Amy
• PICHER, Ángel
• LANCKRIET, HEIKKI

Assignees

• 4basebio UK Ltd

Dates

Publication Date

20260513

Application Date

20241107

Claims (15)

1. A method for producing a protected DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the method comprises: (a) generating a partially protected double-stranded DNA (dsDNA) comprising a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises a cassette, x nuclease-resistant nucleotides at the 5' end of the cassette or 5' of the cassette, and y nuclease-resistant nucleotides at the 3'-end of the cassette or 3' of the cassette, wherein x is at least 1 and y is at least 1, wherein the step of generating the partially protected dsDNA comprises: (i) contacting a precursor dsDNA comprising a first strand and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5' of the cassette and an endonuclease target sequence 3' of the cassette; (ii) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA; (iii) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises x nuclease resistant nucleotides and the second adaptor molecule comprises y nuclease-resistant nucleotides and wherein x is at least 1 and y is at least 1, and wherein the ligase is T7 DNA ligase; and (iv) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating the partially protected dsDNA; and (b) digesting the second strand of the partially protected dsDNA with an exonuclease mixture thereby generating the protected DNA, wherein the exonuclease mixture comprises at least a 5' to 3' exonuclease and a 3' to 5' exonuclease.
2. A method for producing a protected DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the method comprises: (a) providing a partially protected double-stranded DNA (dsDNA) comprising a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises a cassette, x nuclease-resistant nucleotides at the 5' end of the cassette or 5' of the cassette, and y nuclease-resistant nucleotides at the 3'-end of the cassette or 3' of the cassette, wherein x is at least 1 and y is at least 1; and (b) digesting the second strand of the partially protected dsDNA with an exonuclease mixture thereby generating the protected DNA, wherein the exonuclease mixture comprises at least three exonucleases, wherein at least one of the at least three exonucleases is a 5' to 3' exonuclease and at least one of the at least three exonucleases is a 3' to 5' exonuclease.
3. The method of claim 1 or claim 2, wherein the exonuclease mixture comprises at least two 5' to 3' exonucleases.
4. The method of any one of claims 1-3, wherein the exonuclease mixture comprises at least two 3' to 5' exonucleases.
5. The method of any one of claims 1-4, wherein the exonuclease mixture comprises at least four exonucleases, optionally wherein at least two of the at least four exonucleases are 5' to 3' exonucleases and at least two of the at least four exonucleases are 3' to 5' exonucleases.
6. The method of any one of claims 1-5, wherein the 5' to 3' exonuclease is Lambda exonuclease and/or T7 exonuclease.
7. The method of any one of claims 1-6, wherein the 3' to 5' exonuclease is exonuclease I and/or exonuclease III.
8. The method of claim 5, wherein the at least four exonucleases are Lambda exonuclease, T7 exonuclease, exonuclease I, and exonuclease III.
9. The method of claim 2, wherein the method further comprises a step of generating the partially protected dsDNA.
10. The method of claim 9, wherein the step of generating the partially protected dsDNA comprises: (i) contacting a precursor dsDNA comprising a first strand and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5' of the cassette and an endonuclease target sequence 3' of the cassette; (ii) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA; (iii) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises x nuclease resistant nucleotides and the second adaptor molecule comprises y nuclease-resistant nucleotides and wherein x is at least 1 and y is at least 1; and (iv) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating the partially protected dsDNA.
11. The method of claim 1 or claim 10, wherein steps (i)-(iv) are performed in a single contiguous aqueous volume.
12. The method of claim 11, wherein step (b) comprising contacting the single contiguous aqueous volume with the exonuclease mixture without purifying the partially protected dsDNA.
13. The method of claim 10, wherein the ligase is a T7 DNA ligase.
14. The method of any one of claims 1 or 9-13, wherein the method further comprises: amplifying a DNA template to generate the precursor dsDNA, wherein the DNA template comprises the cassette and the endonuclease target sequences, optionally wherein the DNA template is amplified by rolling circle amplification.
15. The method of anyone of claims 1-14, further comprising the step of nicking the second strand of the partially protected dsDNA prior or at the same time as digesting the second strand of the partially protected dsDNA, optionally wherein the nicking is performed by a DNA glycosylase, a nicking endonuclease or an AP endonuclease.

Description

TECHNICAL FIELD The present invention relates to protected DNA comprising a single-stranded DNA (ssDNA) cassette and uses thereof. The present invention also relates to methods for producing a protected DNA, products generated in performing such methods (including intermediate and final products), and kits for use in such methods. BACKGROUND DNA is susceptible to degradation by nucleases which are naturally occurring enzymes within organisms and which have a vital role in the regulation of many cellular processes, while also protecting against foreign DNA species. Enzymatic DNA degradation can render gene therapies ineffective and is a substantial consideration when developing gene therapies or DNA vaccines. Considerable efforts have been made to extend the effective molecular lifetime of nucleic acids by increasing resistance of the nucleic acid molecules to both extracellular and intracellular nucleases. For linear molecules, one of the proposed solutions includes the use of phosphorothioated nucleotides (i.e. 2'-deoxynucleotides-5'-(α-thio)-triphosphate). Phosphorothioated nucleotides comprise a sulphur atom instead of a non-bridging oxygen atom. These modified nucleotides show comparable physical and chemical characteristics to corresponding unmodified nucleotides, but are resistant to exonuclease digestion. As such, the incorporation of the phosphorothioate functional group can prolong the half-life of the nucleic acid molecule. Phosphorothioate modifications have also been used in the context of a linear double-stranded polynucleotide chain (e.g. double-stranded DNA) to cap the ends of the polynucleotide chain to increase resistance to exonuclease digestion (Putney et al. "A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo." Proceedings of the National Academy of Sciences 78.12 (1981): 7350-7354). To cap the ends of the polynucleotide chain, the ends are digested with a restriction enzyme and treated with a DNA polymerase and a mixture of deoxyribonucleotide triphosphates (dNTPs), at least one type of which is a phosphorothioated nucleotide complementary to a nucleotide in the overhanging strand. Since DNA polymerases add nucleotides in the 5' to 3' direction, the result of this treatment is a blunt-ended polynucleotide fragment with a phosphorothioated nucleotide located at the 3'-end of each strand (i.e. in "the cap"). In addition, in therapeutic nucleic acids, phosphorothioated nucleotides are incorporated into short, single-stranded polynucleotide chains. For example, an antisense oligonucleotide fomivirsen is a 21-mer phosphorothioate oligodeoxynucleotide used to treat cytomegalovirus retinitis (Stein and Castanotto, "FDA-approved oligonucleotide therapies in 2017." Molecular Therapy 25.5 (2017): 1069-1075). Similarly, pegaptanib (brand name Macugen) is a short (27-nucleotides) aptamer with a phosphorothioate 3'-3' deoxythymidine cap used for treating age-related macular degeneration of the retina. However, chemical synthesis of such single-stranded polynucleotides is limited to the generation of relatively short nucleotides. Thus, chemical synthesis of longer single-stranded polynucleotides (>100 mer) presents a number of challenges. For example, as the DNA length starts to exceed 100 nucleotides, the yield of desired products becomes limited by side reactions and even modest inefficiencies within the stepwise chemical reactions have large effects. WO2024/017978 describes methods which are capable of generating long single-stranded DNA molecules particularly longer single-stranded DNA molecules which are stable for in vivo applications. In the context of in vivo applications, however, the yield and purity of the synthetic product is crucially important. Thus, a need exists for efficient methods which are capable of generating long single-stranded DNA molecules at high yield and purity. DESCRIPTION The invention provides improved methods for producing a protected DNA comprising a single-stranded DNA (ssDNA) cassette (e.g. a long ssDNA cassette). The methods may generate a higher yield of the protected DNA and/or a higher purity of the protected DNA compared to known methods (e.g. the methods described in WO2024/017978). The inventors have surprisingly discovered that the use of T7 DNA ligase in the methods described herein produces a higher yield of the protected DNA. The inventors have also surprisingly discovered that an exonuclease mixture used during the step of digestion as described herein improves the purity of the protected DNA. Accordingly, the methods of the invention, which are based on these surprising discoveries, may generate protected DNA with a higher yield and a higher purity. The invention provides a method for producing a protected DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the protected DNA comprises a nuclease-resistant nucleotide at the 5' end of the