Method for the generation of single stranded DNA

Abstract

The present invention relates to a method for the generation of single stranded DNA (ssDNA) molecules from circular double stranded DNA (dsDNA) molecules comprising a region of interest.

Description

[0001] Method for the generation of single stranded DNA

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the generation of single stranded DNA (ssDNA) starting from double stranded DNA (dsDNA).

[0004] BACKGROUND

[0005] Gene therapy aims to cure or even prevent diseases by delivering the appropriate genetic information. Adeno-associated viruses (AAV) based vectors have been successfully used as vehicles for the therapeutic nucleic acids. To ensure the efficacy and safety of the therapy, it is essential to control the quality of the active pharmaceutical ingredient, i.e., the nucleic acids. Single stranded AAV DNA is needed as homogenous standard for the development of analytical methods to determine the quality of the produced AAV particles. Additionally, ssDNA can be used for in vitro production of AAV particles. Therefore, there is a need for an in vitro method for the generation of ssDNA.

[0006] SUMMARY

[0007] In a first aspect, the present invention provides a method for the generation of single stranded DNA (ssDNA) molecules from circular double stranded DNA (dsDNA) molecules comprising a region of interest, the method comprising the following steps:

[0008] a) providing circular dsDNA molecules,

[0009] b) cutting the circular dsDNA molecules near one end of the region of interest by a cleavage enzyme resulting in linear dsDNA molecules with sticky ends,

[0010] c) enzymatic fill up of the sticky ends with biotinylated dNTPs,

[0011] d) binding of the biotinylated DNA to magnetic Streptavidin Beads,

[0012] e) cutting the linear dsDNA molecules by a cleavage enzyme near a second end of the region of interest,

[0013] f) washing away the backbone,

[0014] g) denaturing the bound DNA and retrieving the ssDNA comprising the region of interest from the supernatant.

[0015] In an embodiment of the present invention, the one end of the region of interest is the 5’ end of the region of interest and the second end of the region of interest is the 3’ end of the region of interest. In an embodiment of the present invention, the one end of the region of interest is the 3’ end of the region of interest and the second end of the region of interest is the 5’ end of the region of interest.

[0016] In an embodiment of the present invention, the dsDNA in step a) is an AAV plasmid comprising an AAV vector comprising a transgene flanked by one ITR upstream and downstream of the transgene and a plasmid backbone fragment. The AAV vector comprising the transgene flanked by one ITR upstream and downstream of the transgene is the region of interest as defined in this patent application.

[0017] In an embodiment of the present invention, the cleavage enzyme in step e) generates an AAV plasmid backbone fragment and an AAV vector fragment.In an embodiment of the present invention, the cleavage enzyme in step e) cleaves inside or outside the region of interest to generate a ssDNA of the region of interest which is truncated or prolonged compared to the full length region of interest.

[0018] In an embodiment of the present invention, different combinations of biotinylated and non-bioti-nylated dNTPs are used in step c).

[0019] In an embodiment of the present invention, the cleavage enzyme in step b) and in step e) is a restriction enzyme.

[0020] In an embodiment of the present invention, one of the dNTPs in step c) is deoxyuracil triphosphate Uracil (dUTP).

[0021] In an embodiment of the present invention, where in step c) dUTP is one of the dNTPs used, the second cleavage enzyme in step e) is a combination of Uracil-DNA glycosylase and Apurinic / apyrim-idinic endonuclease 1.In an embodiment of the present invention, the DNA in step g) is denatured by a base, preferably NaOH.

[0022] In another embodiment of the present invention, where in step c) dUTP is one of the dNTPs used, the method comprises an additional step h) after step g), wherein in step h) the ssDNA strand which is bound by Streptavidin beads is cleaved by Uracil-DNA-deglycosylase and Apurinic / apyrimidinic Endonuclease land the released ssDNA strand is recovered from the supernatant.

[0023] In a second aspect, the present invention provides a use of the ssDNA produced according to the method of the present invention for the in vitro production of AAV particles.

[0024] In a third aspect, the present invention provides a use of the ssDNA produced according to the method of the present invention as homogenous standard for the development of analytical methods to determine the quality of the produced AAV particles..

[0025] SHORT DESCRIPTION OF THE FIGURES

[0026] Fig. 1 is a graphical presentation of the different steps of the ssDNA generating method of the present invention,

[0027] Fig. 2 shows an agarose gel with the DNA bands generated by the ssDNA generating method using the restriction enzymes PaqCI, PacI and Stul and the fill-up of the resulting ssDNA to dsDNA using a DNA-polymerase .

[0028] Fig. 3A shows the resulting ssDNA using an ssDNA ladder on the Fragment Analyzer.

[0029] Fig. 3B shows the fill-up of the resulting ssDNA to dsDNA using a dsDNA ladder on the Fragment Analyzer.

[0030] DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0031] Useful methods and techniques for carrying out the subject matter of the current invention are described in e.g. Ausubel, F.M. (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, N.D., and Hames, B.D., ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, R.I. (ed.), Animal Cell Culture - a practical approach, IRL Press Limited (1986); Watson, J.D., et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, E.L.,From Genes to Clones; N.Y., VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., N.Y. (1987). The use of recombinant DNA technology enables the generation of derivatives of a nucleic acid. Such derivatives can, for example, be modified in individual or several nucleotide positions by substitution, alteration, exchange, deletion or insertion. The modification or derivati-zation can, for example, be carried out by means of site directed mutagenesis. Such modifications can easily be carried out by a person skilled in the art (see e.g. Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, B.D., and Higgins, S.G., Nucleic acid hybridization - a practical approach (1985) IRL Press, Oxford, England).

[0032] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably.

[0033] The terms “comprise(s)”, “include(s)”, “having”, “has”, “can”, “contain(s)” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude the possibility of additional acts or structures. The term “comprising” also encompasses the term “consisting of’. The current invention also contemplates other embodiments “comprising”, “consisting of’ and “consisting essentially of’ the embodiments or elements presented herein, whether explicitly set forth or not.

[0034] The term “recombinant cell” as used herein denotes a cell after genetic modification, such as, e.g., a cell expressing a heterologous polypeptide of interest and that can be used for the production of said heterologous polypeptide of interest at any scale. For example, “a recombinant mammalian cell comprising an exogenous nucleotide sequence” denotes a cell wherein the coding sequences for a heterologous polypeptide of interest have been introduced into the genome of the host cell. For example, “a recombinant mammalian cell comprising an exogenous nucleotide sequence” that has been subjected to recombinase mediated cassette exchange (RMCE) whereby the coding sequences for a polypeptide of interest have been introduced into the genome of the host cell is a “recombinant cell”.

[0035] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachro-mosomally or at a chromosomal location that is different from its natural chromosomal location.

[0036] The terms "vector" or “plasmid”, which can be used interchangeably, as used herein, refer to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

[0037] The term “nucleic acid molecule” or “polynucleotide” includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- orpyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors, can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or expression of the encoded molecule so that mRNA can be injected into a subject to generate the antibody in vivo (see e.g., Stadler al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or EP 2 101 823 Bl).

[0038] The term “AAV” is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single- stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. There are currently thirteen serotypes of AAV that have been characterized. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, it is fully expected that these same principles will be applicable to additional AAV serotypes since it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3:1-61(1974)).

[0039] An "AAV vector" as used herein refers to a vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs). The transgene of interest is inserted into the AAV genome, replacing the viral genes while retaining the viral inverted terminal repeats (ITRs) that are essential for packaging and replication. The AAV vector backbone is a crucial component of AAV-based gene delivery systems. It refers to the engineered genetic structure that facilitates the packaging and delivery of therapeutic genes into target cells. Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products.

[0040] An "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "AAV vector particle" or simply an "AAV vector".ITR stands for "Inverted Terminal Repeat." In the context of molecular biology and genetics, ITRs are short, repetitive sequences found at the ends of certain DNA molecules, such as viral genomes, especially those of adeno-associated viruses (AAVs). These sequences play crucial roles in the replication, packaging, and integration of viral genomes.

[0041] dNTP stands for "deoxyribonucleotide triphosphate," that serve as the building block for DNA synthesis. Each dNTP consists of three components: a deoxyribose sugar, a nitrogenous base, and three phosphate groups.

[0042] A DNA cleavage enzyme is protein that cut DNA molecules at specific sites.

[0043] A "region of interest "refers to a specific segment of a DNA sequence that is of particular significance for a given study or application. The region of interest might include a specific gene or set of genes that are being studied for their role in a particular biological process or disease.

[0044] EXAMPLE

[0045] Generation of ssAAV DNA from AAV vector plasmids

[0046] Used plasmid mGL-Kanamycin-resistance (Roche ID: D1AK5211)

[0047] 1 , Digest Plasmid with PaqCI (NEB)

[0048] • 1 pl (10 u) PaqCI / pg plasmid DNA

[0049] • 1 pl PaqCI Activator / pg Plasmid DNA

[0050] • lx rcutSmart

[0051] • Fill up with Nuclease free water to 50 pl / pg plasmid DNA

[0052] • Digest: 1 h @ 37°C

[0053] • Heat inactivation: 20 min @ 65°C

[0054] 2, Fill up the sticky ends with Klenow Fragment (3’ — >5’ exo-) (NEB) with biotinylated dNTPs (Thermo Fisher)

[0055] • Add 6 pl (30 u) Klenow Fragment (3’ — >5’ exo-) / pg plasmid DNA

[0056] • IxNEBuffer 2.0

[0057] • Add dTTP, dGTP, biotinylated dATP, biotinylated dCTP to a final concentration of 0.05 mM each

[0058] • Fill up with Nuclease free water to 100 pl / pg plasmid DNA

[0059] • Fill up: 1 h @ 37°C

[0060] 3 , Magnetic Bead Cleanup to remove free biotinylated dNTPs

[0061] • Adjust ProNex® Beads (Promega) to room-temperature

[0062] • Add 200 pl ProNex® Beads / pg plasmid DNA

[0063] • Let the DNA bind to the beads for 15 min @RT

[0064] • Place the tube in a magnetic separation rack and remove the supernatant

[0065] • Wash the beads two times with 80% EtOHRemove the tube from the magnetic separation rack and resuspend the beads in 100 pl Nuclease free water / pg plasmid DNA

[0066] , Wash Dynabeads MyOne Streptavidin Cl Beads (Thermo Fisher)

[0067] • Resuspend the beads

[0068] • Transfer 50 pl beads / pg Plasmid DNA to a tube

[0069] • Wash the beads with 0.7 ml lx Binding & Washing Buffer (5 mM Tris-HCl (pH 8), 0.5 mM EDTA, 1 M NaCl)

[0070] • Place the tube in a magnetic separation rack and remove the supernatant

[0071] • Resuspend the Beads in 50 pl lx Binding & Washing Buffer

[0072] • Repeat the washing steps 2x for a total of 3 washes

[0073] • Resuspend the Beads in 100 pl 2x Binding & Washing Buffer

[0074] , Bind the DNA to the beads

[0075] • Add 100 pl biotinylated DNA to 100 pl of washed Streptavidin Beads

[0076] • On a rotator, let the DNA bind to the beads for 30 min @RT

[0077] • Place the tube in a magnetic separation rack and remove the supernatant

[0078] • Remove the tube from the magnetic separation rack

[0079] • Resuspend the Beads in 250 pl lx Binding & Washing Buffer and incubate it for 10 min on a rotator

[0080] • Place the tube in a magnetic separation rack and remove the supernatant

[0081] • Repeat the washing steps 2x for a total of 3 washes

[0082] , Cut out the plasmid backbone with Stul (NEB) and Pact (NEB)

[0083] • Resuspend the Beads in 48.4 pl Nuclease free water / pg Plasmid DNA

[0084] • Add 6 pl CutSmart

[0085] • Add 2.8 pl (28 u) Stul

[0086] • Add 2.8 pl (28 u) Pact

[0087] • Digest: 1 h @37°C and 400 rpm

[0088] , Wash the beads

[0089] • Place the tube in a magnetic separation rack and remove the supernatant

[0090] • Remove the tube from the magnetic separation rack

[0091] • Resuspend the Beads in 100 pl lx SSC Buffer / pg Plasmid DNA and incubate it for 10 min on a rotator

[0092] • Place the tube in a magnetic separation rack and remove the supernatant

[0093] , Elute the ssDNA

[0094] • Resuspend the Beads in 40 pl 0.15 M NaOH / pg Plasmid NDA

[0095] • Incubate for 10 min on a rotator• Neutralize the mixture by adding 4.4 pl lOx TE buffer (pH 8) and 2.6 pl 1.25 M acetic acid • Place the tube in a magnetic separation rack and save the supernatant containing the non-biotinyl- ated ssDNA strand in a new tube

Claims

CLAIMS1. A method for the generation of single stranded DNA (ssDNA) from circular double stranded DNA (dsDNA) comprising a region of interest, the method comprising the following steps: a) providing circular dsDNA molecules,b) cutting the circular dsDNA molecules near one end of the region of interest by a cleavage enzyme resulting in linear dsDNA molecules with sticky ends,c) enzymatic fdl up of the sticky ends with biotinylated dNTPs,d) binding of the biotinylated DNA to magnetic Streptavidin Beads,e) cutting the linear dsDNA molecules by a cleavage enzyme near a second end of the region of interest,f) washing away the backbone,g) denaturing the bound DNA and recovering the ssDNA comprising the region of interest from the supernatant.

2. The method of claim 1, wherein in the dsDNA in step a) is an AAV plasmid comprising an AAV vector comprising a transgene flanked by one ITR upstream and downstream of the transgene and a plasmid backbone fragment.

3. The method of claim 2, wherein the cleavage enzyme in step e) generates an AAV plasmid backbone fragment and an AAV vector fragment.

4. The method of claim 1 or 2, wherein the cleavage enzyme in step e) cleaves inside or outside the region of interest to generate a ssDNA of the region of interest which is truncated or prolonged compared to the full length of the region of interest.

5. The method of claims 1 - 4, wherein in step c) different combinations of biotinylated and non-biotinylated dNTPs are used.

6. The method of claims 1 - 5, wherein in step b) and in step e) the cleavage enzyme is a restriction enzyme.

7. The method of claims 1 - 6, wherein in step c) one of the dNTPs is deoxyuracil triphosphate Uracil (dUTP).8 The method of claim 7, wherein in step e) the second cleavage enzyme is a combination of a Uracil-DNA-deglycosylase and Apurinic / apyrimidinic Endonuclease 1.

9. The method of claims 1 - 8, wherein in step g) the DNA is denatured by a base, preferably NaOH.

10. The method of claim 7 comprising an additional step h) after step g), wherein in step h) the ssDNA strand which is bound by Streptavidin beads is cleaved by Uracil-DNA-deglycosylase and Apurinic / apyrimidinic Endonuclease land the released ssDNA strand is recovered from the supernatant.

11. Use of the ssDNA produced according to the method of claims 2 - 10 for the in vitro production of AAV particles.

12. Use of the ssDNA produced according to the method of claims 2 - 10 as homogenous standard for the development of analytical methods to determine the quality of produced AAV particles.9

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