Vectors encapsidating nucleic acids, production methods and uses thereof

Abstract

Disclosed herein is a nucleic acid delivery vector comprising capsid proteins or portions thereof from a non-enveloped mammalian virus, wherein the capsid proteins or portions thereof encapsidate a nucleic acid payload and wherein the capsid proteins or portions thereof were expressed and functionally processed in prokaryotic cells. Also disclosed is a method of producing a nucleic acid delivery vector. The method comprises: expressing capsid proteins or portions thereof from a non-enveloped mammalian virus in prokaryotic cells; optionally purifying the expressed capsid proteins or portions thereof from the prokaryotic cells; combining the expressed capsid proteins or portions thereof with a nucleic acid comprising: a nucleic acid payload; and a DNA packaging sequence of the non-enveloped mammalian virus, wherein the capsid proteins or portions thereof self-assemble and encapsidate the nucleic acid payload forming the nucleic acid delivery vector.

Description

VECTORS ENCAPSIDATING NUCLEIC ACIDS, PRODUCTION METHODS AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from US provisional application number 63 / 650134 filed May 21 , 2024, the contents of which are incorporated herein by reference.FIELD

[0002] The present disclosure relates generally to vectors comprising capsid proteins from a nonenveloped mammalian virus for use in delivering nucleic acids to mammalian cells. More particularly, the present disclosure relates to nucleic acid delivery vectors that encapsidate a nucleic acid payload.BACKGROUND

[0003] The delivery of nucleic acids to mammalian cells is important for applications including, for example, gene therapy, gene editing including gene knock-ins, treatment or prevention of disease, vaccination, drug development, diagnosis of disease and monitoring of disease progression. Viral vectors for delivery of nucleic acids have been developed that exhibit high transfection and gene expression efficacy. However, viral vectors exhibit safety concerns such as high immunogenicity, risk of insertional mutagenesis, and the possibility of recombination into replication competent viruses. Viral vectors also exhibit limitations with respect to cargo capacity which together with the immunogenicity limit their efficacy. Therefore, delivery of nucleic acids to cells using viral vectors remains a challenge due to limited packaging size; immune reactions; off-target effects; inflammation; and insertional mutagenesis. There are also challenges with production of viral vectors including scalability issues, difficulties maintaining productivity, and an overall lack of robustness as well as other issues.

[0004] There is a need for novel vectors for delivery of nucleic acids to cells and for nucleic acid delivery systems and methods for delivering deoxyribonucleic acids to cells.SUMMARY

[0005] The inventors have surprisingly found that capsid proteins, such as anellovirus capsid proteins are expressed and functionally processed in prokaryotic cells, such as E. coli cells, and,when mixed with a nucleic acid payload, self-assemble into vectors that encapsidate the nucleic acid payload. The disclosed nucleic acid delivery vectors are useful for delivery of a nucleic acid payload to a target cell. Also disclosed are methods for the assembly and production of vectors.

[0006] Disclosed herein is a nucleic acid delivery vector comprising capsid proteins or a portion thereof from a non-enveloped mammalian virus, wherein the capsid proteins or portions thereof encapsidate a nucleic acid payload and wherein the capsid proteins or portions thereof were expressed and functionally processed in prokaryotic cells.

[0007] In some embodiments of the nucleic acid delivery vector, the prokaryotic cells are E. coli cells. The prokaryotic cells may be protease deficient. The E.coli cells may be BL21 cells.

[0008] In some embodiments of the nucleic acid delivery vector, the nucleic acid payload is a single-stranded deoxyribonucleic acid (ssDNA), a double stranded deoxyribonucleic acid (dsD- NA), or a ribonucleic acid (RNA). The nucleic acid payload may be self-complementary and may be a linear, covalently closed DNA. The nucleic acid payload may be circular. The nucleic acid payload may be a synthetic nucleic acid. The nucleic acid payload may be produced in eukaryotic cells, wherein the eukaryotic cells are yeast or mammalian cells. The nucleic acid payload may be produced in prokaryotic cells.

[0009] In some embodiments of the nucleic acid delivery vector, the nucleic acid payload comprises filamentous phage-processed DNA. The nucleic acid payload may comprise MIS- processed single stranded circular DNA. The nucleic acid payload may comprise a DNA minivector comprising a nucleic acid payload and a filamentous phage origin of replication, wherein the DNA minivector is devoid of prokaryotic vector backbone genetic sequences. The filamentous phage origin of replication may be an M13 origin of replication or an f1 origin of replication.

[0010] In some embodiments of the nucleic acid delivery vector, the non-enveloped mammalian virus is an anellovirus. The anellovirus may be from the genera Alphatorquevirus, Betatorquevirus, Deltatorquevirus, Epsilontorquevirus, Etatorquevirus, Gammatorquevirus, lotatorquevirus, Kappatorquevirus, Lambdatorquevirus, Mutorquevirus, Nutorquevirus, Thetatorquevirus, Zetatorquevirus, Gyrovirus, Chitorquevirus, Omegatorquevirus, Omicrontorquevirus, Pitorquevirus, Psitorquevirus, Rhotorquevirus, Sigmatorquevirus, Upsilontorquevirus, Xitorquevirus, Aleptorquevirus, Dalettorquevirus, Gimeltorquevirus, Hetorquevirus, Tettorquevirus, Wawtorquevirus or Zayintorquevirus.

[0011] In some embodiments of the nucleic acid delivery vector, the capsid protein or portion thereof comprises an anellovirus ORF1 capsid protein or an N-terminal portion of an anellovirus0RF1 capsid protein. The capsid protein or portion thereof may comprise the N-terminal 3rd portion of a TTV19 ORF1 capsid protein, having a molecular weight of 25 kDa.

[0012] In some embodiments of the nucleic acid delivery vector, the capsid protein or portion thereof is encoded by a nucleic acid sequence comprising: the sequence set forth in SEQ ID NO: 1 ; SEQ ID: NO:2; or SEQ ID NO:3; a sequence having at least 80% sequence identity with the sequence set forth in SEQ ID NO:1 ; SEQ ID NO:2; or SEQ ID NO:3; a sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO:1 ; SEQ ID NO:2; or SEQ ID NO:3; a sequence having at least 95% sequence identity with the sequence set forth in SEQ ID NO:1 ; SEQ ID NO:2; or SEQ ID NO:3; or a sequence having at least 99% sequence identity with the sequence set forth in SEQ ID NO:1 ; SEQ ID NO:2; or SEQ ID NO:3. The nucleic acid encoding the capsid proteins or proteins thereof may be codon optimized for E. coli expression. The nucleic acid sequence encoding the capsid proteins or proteins thereof may be CpG dinucleotide- depleted.

[0013] In some embodiments of the nucleic acid delivery vector, the capsid proteins or portions thereof comprise capsid proteins or portions thereof comprising: an amino acid sequence as set forth in SEQ ID NO:4 or SEQ ID NO:5; an amino acid sequence at least 80% identical to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5; an amino acid sequence at least 90% identical to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5; an amino acid sequence at least 95% identical to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5; or an amino acid sequence at least 99% identical to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5; wherein the amino acid sequence at least 80% identical, 90% identical, 95% identical or 99% identical to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5 retains the ability to selfassemble into the nucleic acid delivery vector.

[0014] In some embodiments of the nucleic acid delivery vector, the capsid proteins of portions thereof comprise capsid proteins or portions thereof comprising: an amino acid sequence as set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19; an amino acid sequence at least 80% identical to the sequence set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ I D NO: 17, SEQ I D NO: 18, or SEQ I D NO: 19; an amino acid sequence at least 90% identical to the sequence set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19; an amino acid sequence at least 95%identical to the sequence set forth in SEQ ID N0:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, or SEQ ID NO:19; or an amino acid sequence at least 99% identical to the sequence set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19, wherein the amino acid sequence at least 80% identical, at least 90% identical, at least 95% identical or at least 99% identical to the sequence set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19, retains the ability to selfassemble into the nucleic acid delivery vector.

[0015] In some embodiments of the nucleic acid delivery vector, the capsid proteins or portions thereof comprise an N-terminal tag. The N-terminal tag may be for purifying the capsid proteins or portions thereof and / or for purifying the nucleic acid delivery vector and / or for targeting the nucleic acid delivery vector to a target cell. The N-terminal tag may be a polyhistidine-tag, a FLAG- tag, NE-tag, Strep-tag, Spot-tag, T7-tag, or a TrpE-tag. The tag may be a polyhistidine tag (His- tag).

[0016] In some embodiments of the nucleic acid delivery vector, the capsid proteins or portions thereof were purified from E. coli cells. The capsid proteins may be purified by affinity chromatography. The capsid proteins or portions thereof may be N-terminally His-tagged and are purified by Ni column purification.

[0017] Disclosed herein is a nucleic acid delivery vector comprising capsid proteins or portions thereof from a non-enveloped mammalian virus, wherein the capsid proteins or portions thereof encapsidate a nucleic acid payload and wherein the capsid proteins or portions thereof comprise an N-terminal portion of the TTV19 ORF1 capsid protein that has a molecular weight of approximately 25 kDa. The N-terminal portion of the TTV19 ORF1 capsid protein may encapsidate a synthetic or modified circular ssDNA genome encoding a therapeutic or reporter transgene.

[0018] Disclosed herein is a method of producing a nucleic acid delivery vector, the method comprising: expressing capsid proteins or portions thereof from a non-enveloped mammalian virus in prokaryotic cells; combining the expressed capsid proteins or portions thereof with a nucleic acid comprising: a nucleic acid payload; and a DNA packaging sequence of the nonenveloped mammalian virus, wherein the capsid proteins or portions thereof self-assemble and encapsidate the nucleic acid payload forming the nucleic acid delivery vector.

[0019] In some embodiments of the method of producing a nucleic acid delivery vector, the capsid proteins or portions thereof and the nucleic acid are combined in a 6:1 mass ratio (w / w) of capsid protein or portion thereof to nucleic acid. The DNA packaging sequence may comprise the nucleic acid as set forth in SEQ ID NO: 20, SEQ ID NO:21 or SEQ ID NO:22. The capsid proteins or portions thereof may be N-terminally tagged and the purifying may be done by affinity chromatography. The capsid proteins or portions thereof may be N-terminally His-tagged and are purified by Ni column purification. The method may further comprise a step of purifying the capsid proteins or portions thereof before the step of combining.

[0020] In some embodiments of the method of producing a nucleic acid delivery vector, the step of combining occurs in vitro. The nucleic acid comprising the nucleic acid payload and the DNA packaging sequence of the non-enveloped mammalian virus may be expressed in different prokaryotic cells than the capsid proteins or portions thereof. The capsid proteins or portions thereof and the nucleic acid comprising the nucleic acid payload and the DNA packaging sequence of the non-enveloped mammalian virus may be combined in 1 * assembly buffer solution comprising 50 mM Tris-HCI, 50 mM NaCI, 10 mM KCI and 5 mM MgCh with a pH of 7.2. The step of combining may include an overnight incubation at 4 degrees Celsius.

[0021] In some embodiments of the method of producing a nucleic acid delivery vector, the step of combining occurs in prokaryotic cells and the nucleic acid payload is encapsidated by the capsid proteins or portions thereof in vivo in prokaryotic cells.

[0022] In some embodiments of the method of producing a nucleic acid delivery vector, the method may further comprise a step of purifying the nucleic acid delivery vector. The prokaryotic cells may be E.coli cells. The capsid protein or portion thereof may comprise the N-terminal 3rd portion of a TTV19 ORF1 capsid protein having a molecular weight of 25 kDa. The DNA packaging sequence may be the intergenic region of a TTV19 anellovirus. The DNA packaging sequence may comprise the nucleic acid as set forth in SEQ ID NO: 20, SEQ ID NO:21 or SEQ ID NO:22

[0023] Disclosed herein is a method of producing a nucleic acid delivery vector, the method comprising: co-transforming prokaryotic cells with a nucleic acid sequence encoding a capsid protein or portion thereof and a nucleic acid encoding a nucleic acid payload and a DNA packaging sequence; assembling nucleic acid delivery vectors encapsidating the nucleic acid payload in the prokaryotic cells; and optionally, purifying assembled nucleic acid delivery vectors encapsidating the nucleic acid payload.

[0024] Disclosed herein is a method of producing a TTV19 nucleic acid delivery vector that yields >1O10viral particles / mL at the harvest stage, comprising: expressing TTV-19 capsid proteins or portions thereof from a non-enveloped mammalian virus in prokaryotic cells; combining theexpressed capsid proteins or portions thereof with a nucleic acid comprising: a nucleic acid payload; and a DNA packaging sequence of the TTV19 virus, wherein the TTV19 capsid proteins or portions thereof self-assemble and encapsidate the nucleic acid payload forming the nucleic acid delivery vector.

[0025] In some embodiments of the method of producing a TTV19 nucleic acid delivery vector that yields >1O10viral particles / mL at the harvest stage, the step of combining occurs in vitro and the method may further comprise an optional step of purifying the TTV19 capsid proteins or portions thereof prior to combining them with the nucleic acid. Alternatively, the step of combining may occur in vivo, wherein the nucleic acid is expressed in the same prokaryotic cells as the TTV19 capsid proteins or portions thereof. The method may comprise a further step of purifying the nucleic acid delivery vector.

[0026] Also disclosed are nucleic acid delivery vectors produced by the methods disclosed herein.

[0027] Also disclosed is a cell line or helper system that supports the assembly and replication in vitro of a nucleic acid delivery vector disclosed herein.

[0028] Also disclosed are pharmaceutical compositions comprising the nucleic acid delivery vector in a pharmaceutically acceptable carrier or diluent. The pharmaceutical compositions comprising the nucleic acid delivery vector may be acceptable for therapeutic administration.

[0029] Also disclosed is a scalable process to purify and isolate nucleic acid delivery vectors disclosed herein using rapid-sequential chromatography, wherein the isolated nucleic acid delivery vectors are high-titer and endotoxin free.

[0030] Also disclosed are uses of the nucleic acid delivery vectors disclosed herein or a pharmaceutical composition with the nucleic acid delivery vectors disclosed herein for delivery of the nucleic acid payload to a cell; for gene therapy; for immune-stealth gene delivery to hematopoietic, hepatic, pulmonary, ocular or mucosal tissues; or for repeat-dose systemic or localized gene therapy without eliciting adaptive immune neutralization.

[0031] Also disclosed is a kit for producing a vector, the kit comprising: a DNA vector plasmid comprising: a viral DNA binding sequence; and a nucleic acid payload, wherein the viral DNA binding sequence and the nucleic acid payload are flanked by an M13 f1 origin of replication START sequence and an M13 f1 origin of replication STOP sequence; a helper plasmid for producing and packaging single stranded DNA containing the nucleic acid payload, the helper plasmid comprising all the genes required for M 13 replication, transcription and packaging; and a plasmid for expressing ORF1 capsid proteins or portions thereof in prokaryotic cells.

[0032] Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized. Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0034] Figure 1 depicts a map of an anellovirus genome;

[0035] Figure 2 depicts a schematic of an embodiment of a method of producing a nucleic acid delivery vector as described herein;

[0036] Figure 3 depicts a schematic of a further embodiment of a method of producing a nucleic acid delivery vector as described herein;

[0037] Figure 4 depicts a schematic of an exemplary method of producing a DNA minivector;

[0038] Figure 5 is a schematic of an embodiment of a method of producing a nucleic acid delivery vector as described herein, wherein the nucleic acid payload comprises a DNA minivector;

[0039] Figure 6 is a plasmid map of an exemplary capsid protein expression plasmid;

[0040] Figure 7, Panel A depicts a split origin of replication flanking a nucleic acid payload; Panel B depicts an exemplary DNA vector comprising a nucleic acid payload and an exemplary circular ssDNA after M13 processing;

[0041] Figure 8 is a plasmid map of an exemplary helper phage;

[0042] Figure 9 shows panel of a production of TTV19 Capsid protein;

[0043] Figure 10 Panel A depicts a schematic of TTV19 capsid protein production, extraction and analysis of the crude extract by SDS-PAGE and Western Blot; Panel B shows detection of TTV19 capsid proteins by SDS-PAGE and Western Blot;

[0044] Figure 11 shows a panel showing purification of His-tagged anellovirus capsid protein using a Ni column in Panels A-D;

[0045] Figure 12 shows SEM images of in vitro and in vivo assembled anellomids in Panels A - C;

[0046] Figure 13 shows STEM images of in vitro assembled anellomids in Panels A-B;

[0047] Figure 14 shows high resolution TEM images of in vivo assembled anellomids;

[0048] Figure 15 depicts a plasmid map of an exemplary DNA vector comprising a nucleic acid payload;

[0049] Figure 16 is a graph showing the concentration of in vitro assembled anellovirus vectors;

[0050] Figure 17 is a graph showing the transfection of HEK 293T cells by assembled anellovirus vectors carrying a luciferase gene expression cassette;

[0051] Figure 18 is a schematic of an embodiment of a method of producing a nucleic acid delivery vector in vivo in a prokaryotic cell as described herein;

[0052] Figure 19 is a plasmid map of an exemplary capsid protein expression plasmid;

[0053] Figure 20 is a plasmid map of an exemplary capsid protein expression plasmid;

[0054] Figure 21 is a graph showing the concentration of in vitro and in vivo assembled anellomids;

[0055] Figure 22 is a graph showing that no helper DNA vector plasmid contamination was found in in vivo assembled anellomids;

[0056] Figure 23 is a graph showing the qPCR amplification plot and melt curve detecting no helper plasmid contamination in in vivo assembled anellomids;

[0057] Figure 24 is a panel showing the detection of His-tagged anellomids assembled in vivo by Western blot; and

[0058] Figure 25 is a panel showing the detection of His-tagged anellomids assembled in vivo by Western blot.DETAILED DESCRIPTION

[0059] Disclosed herein is a nucleic acid delivery vector for delivering a nucleic acid payload to a target cell. The nucleic acid delivery vector may comprise capsid proteins or a portion thereof from a non-enveloped mammalian virus that have been expressed and functionally processed in prokaryotic cells. When combined with a nucleic acid comprising a nucleic acid payload and a viral DNA packaging sequence, the capsid proteins self-assemble and encapsidate the nucleic acid payload to form the nucleic acid delivery vector. The step of combining may occur in vitro or in prokaryotic cells.

[0060] The inventors have found that expressing ORF1 proteins or portions thereof in E. coli generates capsid proteins that will self-assemble and encapsidate a nucleic acid payload. It was not expected that prokaryotic cells, such as E. coli cells, could be used to express mammalian viral proteins since prokaryotic cells are not capable of post translational modifications that aregenerally required for eukaryotic proteins. In addition, it was surprisingly found that the contents of the bacterial system do not hinder vector assembly.

[0061] The nucleic acid encapsulated within the proteinaceous exterior formed by the capsid proteins comprises a viral DNA packaging sequence and a nucleic acid payload. The term “payload” is used interchangeably herein with “nucleic acid payload”. The nucleic acid payload may be a target gene of interest (GOI).

[0062] Non-enveloped mammalian viruses comprise a viral genome (DNA or RNA) and viral- coded capsid proteins that surround the genome and are characterized by an absence of a lipid bilayer on the outer part of the virus. Examples of non-enveloped viruses include, but are not limited to, adenovirus, adeno-associated virus (AAV), human papillomavirus (HPV). Anelloviruses are ssDNA viruses and include the genera Alphatorquevirus, Betatorquevirus, Deltatorquevirus, Epsilontorquevirus, Etatorquevirus, Gammatorquevirus, lotatorquevirus, Kappatorquevirus, Lambdatorquevirus, Mutorquevirus, Nutorquevirus, Thetatorquevirus, Zetatorquevirus, Gyrovirus, Chitorquevirus, Omegatorquevirus, Omicrontorquevirus, Pitorquevirus, Psitorquevirus, Rhotorquevirus, Sigmatorquevirus, Upsilontorquevirus, Xitorquevirus, Aleptorquevirus, Dalettorquevirus, Gimeltorquevirus, Hetorquevirus, Tettorquevirus, Wawtorquevirus, Zayintorquevirus, Givrovirus and unclassified anelloviridae.

[0063] Anelloviruses are known as orphan viruses as they are not specifically associated with disease and are commensal microbes. Anelloviruses are omnipresent in the human population and show tropism towards various cells and tissues. Anelloviruses also have high infectivity, are less susceptible to immune response and are amenable to redosing without triggering strong neutralizing antibody responses. There were several challenges in the field for using anellovirus as a vector for gene therapy. First, there were no robust in vitro culture systems or animal models present to propagate the virus. Current manufacturing of the virus is inefficient and demanding and the life cycle and mechanisms of pathogenesis are poorly understood.

[0064] The genome of anelloviruses is composed of a single-stranded negative-sense circular DNA (ssDNA) which ranges from about 2kb to about 4kb. For example, Torque teno virus (TTV) from the genera Alphatorquevirus has a genome size of 3.7-3.8 kb, Torque teno mini virus (TTMV) of the genera Betatorquevirus has a genome size of 2.8-2.9 kb and Torque teno midi virus (TTMDV) of the genera Gammatorquevirus: has a genome size of 3.2 kb. Anelloviruses replicate through a rolling-circle replication mechanism.

[0065] As shown in Figure 1 , the anelloviral genome contains overlapping open reading frames (ORFs) and an untranslated region (UTR). Within the UTR is an intergenic region which comprises a DNA packaging sequence. The DNA packaging sequence is a nucleotide sequence that bindsDNA binding proteins that are involved in packaging DNA. The DNA packaging sequence is often AT rich.

[0066] Six types of proteins are produced upon translation: ORF1 protein; ORF2 protein; ORF 2 / 2 and ORF 2 / 3 proteins; and ORF 1 / 1 and ORF 1 / 2 proteins. The anelloviral ORF1 proteins are involved in capsid production and are required for packaging DNA (Sarker et al., 2016). ORF1 has a sequence of arginine repeats at its N-terminal end (Arg-rich region) that is similar to the ARM motif discovered in the Cap proteins of circoviruses, which is known to have DNA binding properties. ORF2 proteins are regulatory proteins, ORF 2 / 2 and ORF2 / 3 proteins are localized in nucleus and ORF 1 / 1 and ORF1 / 2 proteins are equally present in nucleus and cytoplasm

[0067] Genomic characteristics and locations of transcription elements of exemplary Anellovirus species are outlined in Table 1.

[0068] Also disclosed herein is a method of producing a nucleic acid delivery vector. In an embodiment, the method comprises: expressing capsid proteins or portions thereof from a nonenveloped mammalian virus in prokaryotic cells; optionally purifying the expressed capsid proteins or portions thereof from the prokaryotic cells; combining the expressed capsid proteins or portions thereof with a nucleic acid comprising the nucleic acid payload and a DNA packaging sequence of the non-enveloped mammalian virus, wherein the capsid proteins or portions thereof self-assemble and encapsidate the nucleic acid payload forming the nucleic acid delivery vector. The assembled nucleic acid delivery vector comprises capsid proteins or portions thereof in the form of an icosahedral head. When the vector comprises anellovirus capsid proteins or portions thereof, the vector is about 30-50 nm in size. In some embodiments of the present invention, the prokaryotic cells used in this method of producing a nucleic acid delivery vector are E. coli cells.

[0069] Figure 2 depicts a method of producing a nucleic acid delivery vector that encapsidates a payload nucleic acid, wherein the nucleic acid delivery vector is assembled in vitro. This method is referred to herein as “a two-step method”. The capsid proteins or portions thereof are expressed and functionally processed in prokaryotic cells. The nucleic acid comprising the payload nucleic acid and the DNA packaging sequence is combined with the capsid proteins or portions thereof in vitro and the capsid proteins or portions thereof self-assemble to encapsidate the nucleic acid payload. In some embodiments the step of combining comprises adding the nucleic acid comprising the DNA binding sequence and the nucleic acid payload to the prokaryotic cell extract comprising capsid proteins or portions thereof. In some embodiments, the capsid proteins or portions thereof may be purified, followed by the step of combining with the nucleic acid comprising the nucleic acid payload and the DNA binding sequence.

[0070] Figure 3 depicts a method of producing a nucleic acid delivery vector, wherein the nucleic acid delivery vector is assembled in a prokaryotic cell. This method is referred to herein as a “one step” method and the step of “combining” occurs in prokaryotic cells. In the one step method, the capsid proteins or portions thereof and the nucleic acid comprising the nucleic acid payload and the DNA packaging sequence are co-expressed in prokaryotic cells and the nucleic acid delivery vectors self-assemble in the prokaryotic cells. Following self-assembly of the nucleic acid delivery vectors, the prokaryotic cells may be lysed. The method may include a step of purification of the assembled nucleic acid delivery vectors to separate the assembled nucleic acid vectors from the prokaryotic cell lysate.

[0071] In an embodiment, there is provided a method of producing a nucleic acid delivery vector comprising: co-transforming prokaryotic cells with a nucleic acid encoding a capsid protein sequence or a portion thereof and a nucleic acid encoding a nucleic acid payload and a DNApackaging signal; assembling nucleic acid delivery vectors encapsidating the nucleic acid payload in the prokaryotic cells; and optionally, purifying assembled nucleic acid delivery vectors encapsidating the nucleic acid payload.

[0072] Any suitable prokaryotic cells may be used in the methods disclosed herein. In an embodiment, the prokaryotic cells are E coli cells. In some embodiments, the E. coli cells are protease deficient (or resistant). Examples of protease deficient E. coli cells include BL21 cells, XL-1 Blue, JM109 and SI4. The person of skill in the art would know what prokaryotic cell strains would be suitable for use in the methods disclosed herein.

[0073] When the step of combining occurs in vitro, the capsid proteins or portions thereof and the nucleic acid comprising the nucleic acid payload and the DNA packaging sequence of the nonenveloped mammalian virus may be combined in an assembly buffer solution. In an embodiment, the assembly buffer comprises 50 mM Tris-HCI, 50 mM NaCI, 10 mM KCI and 5 mM MgCh with a pH of 7.2. When the step of combining occurs in vivo in prokaryotic cells, the nucleic acid payload is encapsidated by the capsid proteins or portions thereof in vivo and no assembly buffer is required. Upon combination, the capsid proteins or portions thereof self-assemble and encapsidate the nucleic acid payload forming the nucleic acid delivery vector. In an embodiment, the capsid proteins or portions thereof and the ssDNA are combined in a 6:1 mass ratio (w / w) of capsid protein or portion thereof to payload nucleic acid.

[0074] The step of combining may include an incubation between about 37 degrees Celsius and about 4 degrees Celsius. In an embodiment the incubation may be at room temperature. In an embodiment, the incubation may be at about 4 degrees Celsius. In an embodiment the step of combining may comprise an overnight incubation. The overnight incubation may be done at about 4 degrees Celsius.

[0075] In an embodiment, the capsid protein or portion thereof comprises an anellovirus ORF1 capsid protein. Tables 1 and 2 provide locus information of exemplary anelloviruses, amino acid sequences of exemplary anellovirus capsid proteins and exemplary nucleic acids encoding ORF1 capsid proteins or portions thereof. In an embodiment, the capsid protein of portion thereof is the N-terminal portion of the TTV19 ORF1 capsid protein that has a molecular weight of approximately 25 kDa. The N-terminal portion of TTV19 ORF1 capsid protein is able to encapsidate a nucleic acid payload in vitro and in vivo in prokaryotic cells.

[0076] In an embodiment, the capsid proteins or portions thereof comprise capsid proteins or portions thereof comprising: an amino acid sequence as set forth in SEQ ID NO:4 or SEQ ID NO:5; an amino acid sequence at least 80% identical to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5; an amino acid sequence at least 90% identical to the sequence set forth in SEQID N0:4 or SEQ ID NO:5; an amino acid sequence at least 95% identical to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5; or an amino acid sequence at least 99% identical to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5;

[0077] wherein the amino acid sequence at least 80% identical, 90% identical, 95% identical or 99% identical to the sequence set forth in SEQ I D NO:4 or SEQ I D NO:5 retains the ability to selfassemble into the nucleic acid delivery vector.

[0078] In an embodiment, the capsid proteins or portions thereof comprise capsid proteins or portions thereof comprising: an amino acid sequence as set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19;an amino acid sequence at least 80% identical to the sequence set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19;an amino acid sequence at least 90% identical to the sequence set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18, or SEQ ID NO: 19; an amino acid sequence at least 95% identical to the sequence set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19; or an amino acid sequence at least 99% identical to the sequence set forth in SEQID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19, wherein the amino acid sequence at least 80% identical, at least 90% identical, at least 95% identical or at least 99% identical to the sequence set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19, retains the ability to self-assemble into the nucleic acid delivery vector.

[0079] In an embodiment, the nucleic acid packaging sequence comprises the nucleic acid as set forth in SEQ ID NO: 20, SEQ ID NO:21 or SEQ ID NO:22.

[0080] In an embodiment, the nucleic acid sequence encoding the capsid protein or portions thereof has been optimized for protein production in bacteria.

[0081] Minimal Capsid protein

[0082] The inventors have surprisingly found that ORF1 capsid proteins when expressed in prokaryotic cells are processed into a smaller form that retains the ability to self-assemble into vectors. This minimal capsid protein, also referred to as a portion of a capsid protein, comprises N-terminal amino acids of the ORF1 capsid protein and have the ability to self-assemble into a vector and to encapsidate a nucleic acid. In an embodiment, the minimal capsid protein is about 20kDa to about 30 kDa. In an embodiment, the capsid protein is about 25kDa. In an embodiment, the minimal capsid protein comprises approximately one third of the N terminal amino acid residues of the ORF1 protein sequence. Because the smaller form (minimal capsid protein) of the capsid proteins comprises the N-terminal amino acids, the smaller form may be purified using an N-terminal tag or an antibody specific to the N-terminus of the capsid protein. The smaller, minimal capsid proteins may be purified from prokaryotic cells by affinity purification. In an embodiment, the capsid protein comprises the amino acids as set forth in SEQ ID NO: 19.

[0083] In an embodiment, the capsid protein or portion thereof comprises an amino acid sequence as set forth in SEQ ID NO:5 or an amino acid sequence at least 80%, 90%, 95% or 99% identical to the sequence set forth in SEQ ID NO:3, along the length of the molecule. In an embodiment, the capsid proteins or portions thereof are about 25 kDa in size and comprise the N-terminal amino acids of the ORF1 capsid protein.

[0084] In an embodiment the nucleic acid delivery vector comprises homomultimeric capsid proteins or portions thereof. For example, the nucleic acid delivery vector comprises only capsid proteins or portions thereof comprising the smaller or “minimal” capsid protein. In an embodiment the nucleic acid delivery vector comprises heteromultimeric capsid proteins or portions thereof. For example, the capsid proteins may comprise a combination of larger or “full’ length capsid proteins and minimal capsid proteins.

[0085] Protein Tags

[0086] The inventors have surprisingly found that incorporating a protein tag at the N-terminus of the capsid protein or portion thereof does not interfere with capsid expression and / or selfassembly of the capsid proteins or portions thereof into vectors. In some embodiments the capsid proteins or portions thereof comprise an N-terminal tag. Examples of suitable tags would be known to the person of skill in the art and may include a polyhistidine-tag (His-tag), a FLAG-tag, a NE-tag, a Strep-tag, a Spot-tag, a T7-tag, or a TrpE-tags. Tags may be used to purify the capsid proteins or portions thereof, for example, by affinity chromatography, such as by nickel column purification. Purification techniques and protocols for purifying proteins would be known to the person of skill in the art. The capsid proteins may be purified from prokaryotic cells prior toassembly into vectors. The fully assembled nucleic acid delivery vectors may be purified using the N-terminal tag. Tags may be used to identify capsid proteins or portions thereof. The capsid protein or minimal capsid protein may be tagged at the N-terminus to allow for purification. The N-terminal tag may be a protein tag and may be, for example, a polyhistidine-tag (His-tag), a FLAG-tag, NE-tag, Strep-tag, Spot-tag, T7-tag, or a TrpE-tag. N-terminally-tagged capsid proteins or portions thereof may be purified using techniques known in the art, including affinity chromatography. An example of a method purifying proteins include Ni column purification. Suitable tags and purification methods are known to the person of skill in the art. At the time of writing, there were no anellovirus-specific antibodies commercially available. However, should antibodies to the capsid protein be identified, they could be used to purify a capsid protein or minimal capsid protein.

[0087] The step of purifying may be done by affinity chromatography. For example, when the capsid proteins or portions thereof are N-terminally His-tagged, the capsid proteins or portions thereof may be purified by Ni column purification.

[0088] N-terminal tags may also be used to target the vectors comprising capsid proteins or portions thereof to a target cell. The N-terminal protein tag may be a molecule that binds a receptor on a target cell. The tag may be used to bind specifically to a target cell. The N-terminal tag may be used to target the vector to a target cell. For example, the N-terminal tag may be an epidermal growth factor or portion thereof, that may be used to target the vector to a cell expressing the epidermal growth factor receptor. A portion of the epidermal growth factor may be a display peptide EGF. N-terminal tags may be linked to the capsid protein or portion thereof by a flexible linker i.e. (Gly-Gly-Gly-Ser).

[0089] A cleavage site may be present following the N-terminal tag, to allow cleavage of the tag following expression and / or purification of the capsid protein.

[0090] Nucleic Acids

[0091] The nucleic acid that is to be packaged comprises the payload nucleic acid and a DNA packaging sequence. A viral DNA packaging sequence is the region of viral genome responsible for directing packaging of DNA into a virus and may also be referred to as the packaging sequence or signal, or the encapsidation sequence. The viral DNA packaging signal is required for encapsidation of the nucleic acid payload within the capsid proteins or portions thereof forming the nucleic acid delivery vector. In non-enveloped mammalian bacteriophage, the packaging signal is provided in the intergenic region. The intergenic region is the untranslated portion of the genome that contains the DNA packaging signal for the capsid proteins. The DNA packagingsequence is found within the intergenic region of the anellovirus genome and examples of intergenic regions are set forth in Table 1 . For example, the nucleotide sequence of the anellovirus TTV19 intergenic region, spans nucleotides 3697-3808 of AB 025946.

[0092] The nucleic acid payload (or payload) is enclosed within the proteinaceous exterior formed by the capsid proteins or portions thereof. The nucleic acid payload may be any nucleic acid that is suitable to be delivered to a target cell. The nucleic acid payload may be used for any biological purpose that would benefit from the delivery to a cell. The nucleic acid payload could be, for example, a gene for use in gene therapy, immunotherapy, or raising an antibody response. The nucleic acid payload may comprise a DNA vaccine and the delivery of the nucleic acid payload may be useful for vaccination. In another example, the nucleic acid payload may comprise or encode an imaging agent or a diagnostic agent and may be used, for example, in the diagnosis of disease or in monitoring disease progression. The delivery of a nucleic acid payload to a mammalian cell may be useful for the treatment or prevention of a disease or disorder.

[0093] The nucleic acid and payload may be a single-stranded deoxyribonucleic acid (ssDNA) or a double stranded deoxyribonucleic acid (dsDNA) or a ribonucleic acid. In some embodiments, the nucleic acid is circular. The nucleic acid and payload may be self-complementary DNA so the circular ssDNA becomes a linear covalently closed (LCC) DNA. The nucleic acid and payload may be a synthetic nucleic acid and may be synthesized in vitro. The nucleic acid and payload may be produced in eukaryotic cells, for example, yeast or mammalian cells. The nucleic acid and payload may be synthesized in prokaryotic cells, for example, E. coli. The nucleic acid and payload may be DNA that is processed in filamentous phage, for example, an M13 filamentous phage.

[0094] The upper limit of nucleic acid that may be packaged will be determined by the packaging capacity of the capsid proteins or portions thereof, which may vary depending on the type of capsid proteins or portions thereof. When the capsid proteins are anellovirus ORF1 proteins, the packaging capacity is approximately 5000kb. The number of nucleotides that can be packaged will depend on whether the nucleic acid is single stranded or double stranded. The packaging capacity of the vector remains constant, so when the nucleic acid is dsDNA, the maximum size of the DNA to be packaged is half that of a ssDNA target nucleic acid.

[0095] The nucleic acid may comprise a nucleic acid sequence of, for example, between 300- 5000 nucleotides, between 300-4000 nucleotides, between 300-3800 nucleotides, between 300- 3500 nucleotides, between 300-3000 nucleotides, between 300- 2500 nucleotides, between 300- 2000 nucleotides, or between 300-1500 nucleotides. In some embodiments, the nucleic acid comprises a nucleic acid sequence of at least 300 nucleotides, 500 nucleotides, 1000 nucleotides,1500 nucleotides, 2000 nucleotides, 2500 nucleotides, 3000 nucleotides, 4000 nucleotides, 5000 nucleotides, or more.

[0096] In an embodiment, the nucleic acid and payload comprises M13-processed single stranded circular DNA or double stranded DNA. In an embodiment, the nucleic acid and payload is made in an E. coli system that is based on a miniphagemid system (iPhAGEs) of production as outlined in PCT / CA 2023 / 050586 and Wong et al., 2022, Wong et al., 2023, which are incorporated by reference herein. The miniphagemid system capitalizes on filamentous (Ff) phages such as M13 phages to produce DNA minivectors. In an embodiment, the nucleic acid comprises a DNA minivector. DNA Minivectors comprise a filamentous phage origin of replication, a DNA packaging sequence and a nucleic acid payload.

[0097] Figure 4 depicts a method of producing an M13 DNA minivector. The M13 phage replication system comprises a helper plasmid and a DNA vector plasmid. The helper plasmid encodes proteins necessary for M13 phage replication, transcription, and assembly of M13 phagemids intracellularly in bacterial systems. An exemplary helper plasmid is M13SW8 (Wong et al, 2022). The DNA vector plasmid comprises the nucleic acid to be packaged by the filamentous phage (i.e. a viral intergenic region comprising the viral DNA packaging sequence and the nucleic acid payload). The DNA vector plasmid comprises a “split origin of replication” wherein the nucleic acid (including the viral DNA packaging signal and the payload) is flanked between the initiation and termination regions of a phage origin of replication. Separation of the initiation and termination signals of the origin of replication to form the “split origin of replication” results in the replication of only the DNA between these signals and results in minivectors which lack a prokaryotic backbone. Prokaryotic vector backbone genetic sequences include, but are not limited to, genetic elements that are present in plasmids and are required for amplification and maintenance of the plasmid in a bacterial host. Prokaryotic vector backbone sequences are rich in unmethylated cytosine-guanine dinucleotide (CpG) motifs that are known to inhibit transgene expression in mammalian cells due in part to the inflammatory response they induce in cells. Minivectors which lack prokaryotic backbone sequences lack the unmethylated CpG motifs found in bacterial sequences, and therefore escape endosomal degradation and avoid CpG-mediated gene silencing. Prokaryotic vector backbone sequences also contain antibiotic resistance markers that can disseminate antibiotic resistance to the environment. Minivectors lack bacterial antibiotic markers and, accordingly, do not promote antibiotic resistance.

[0098] In the method outlined in Figure 4, a DNA vector plasmid is transformed into an E. coli host that is capable of propagating filamentous phage. The DNA vector plasmid comprises a nucleic acid payload and a DNA packaging sequence flanked between initiation and terminationelements of a Ff origin of replication. The transformed E. coli is infected with a helper phage. A cell line or helper system can support the assembly and replication in vitro of a nucleic acid delivery vector. The bacterial host cells are cultured under conditions that promote replication and amplification of the DNA from the DNA vector plasmid, packaging of the resulting ssDNA into progeny phage and extrusion of the progeny phage from the host cell. Extruded progeny filamentous phage comprising the ssDNA (“M13 phagemid” or“miniphagemid”) are harvested and the ssDNA is extracted and / or purified.

[0099] The DNA minivector may be a ssDNA or a sense-antisense (SAS) minivector. Sense antisense minivector precursors comprise both the sense and antisense sequences of a nucleic acid payload, flanked by separated initiation and termination domains of the f1 functional origin (f1 ori). Insertion of a linker (spacer region) between the sense and antisense sequence stabilizes the molecule during cloning of the constructs. The SAS minivector precursor provides a LCC double-stranded minivector.

[0100] Figure 5 shows an embodiment of a method of producing a nucleic acid delivery vector as described herein, wherein the nucleic acid payload comprises a ssDNA minivector. On the left side is shown the extraction of ssDNA from an M13 phagemid. ssDNA encoding a transgene cistron and packaging signal for anellovirus may be produced and purified as M13 miniphagemids from host E. coli. On the right is shown that His-tagged anellovirus capsid proteins or portions thereof can be expressed and processed in E. coli. M13 processed ssDNA is combined with capsid proteins or portions thereof expressed and purified in E.coli and the capsid proteins or portions thereof self-assemble and encapsidate the ssDNA.

[0101] In an embodiment there is disclosed a method of producing a TTV19 nucleic acid delivery vector that yields >1O10viral particles / mL at the harvest stage.

[0102] DNA encoding capsid proteins

[0103] In an embodiment, the capsid protein or portion thereof is encoded by a nucleic acid sequence comprising: the sequence set forth in SEQ ID NO: 1 ; SEQ ID: NO:2; or SEQ ID NO:3; a sequence having at least 80% sequence identity with the sequence set forth in SEQ ID NO:1 ; SEQ ID NO:2; or SEQ ID NO:3; a sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO:1 ; SEQ ID NO:2; or SEQ ID NO:3;a sequence having at least 95% sequence identity with the sequence set forth in SEQ ID NO:1 ; SEQ ID NO:2; or SEQ ID NO:3; or a sequence having at least 99% sequence identity with the sequence set forth in SEQ ID NO:1 ; SEQ ID NO:2; or SEQ ID NO:3.

[0104] As the nucleic acid sequences disclosed herein may be expressed in E. coli, the nucleic acid sequences may be codon optimized for E. coli expression. For example, codons for each amino acid in the nucleic acid sequence encoding the capsid proteins or portions thereof may be optimized to more closely match those frequently used by the host E. coli. The person of skill in the art would understand how to optimize the codon usage for E. coli, and there are many tools available to the person of skill in the art to aid in generating a codon optimized sequence for E. coli.

[0105] Nucleic acid sequences disclosed herein may be CpG-dinucleotide depleted. By CpG- dinucleotide depleted it is meant that the number of CpG dinucleotides is reduced in the nucleic acid sequence compared to the wild type sequence. In an embodiment, the nucleic acid is substantially depleted of CpG dinucleotides.

[0106] Compositions

[0107] In some embodiments, the vectors disclosed herein can be present in a composition or a pharmaceutical composition. A pharmaceutical composition may comprise a pharmaceutically acceptable carrier or diluent. The term pharmaceutically acceptable carrier refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material involved in carrying or transporting any subject composition or component thereof. Each carrier must be acceptable in the sense of being compatible with the subject composition and its components and not injurious to the subject. Carriers and diluents will be familiar to a person of skill in the art.

[0108] Pharmaceutical compositions are generally formulated to be compatible with the intended method or route of administration; exemplary routes of administration include without limitation oral administration, administration by inhalation, or parenteral (for example, intramuscular, intravenous, subcutaneous, intraperitoneal, intrathecal, or intraarticular). In some embodiments the pharmaceutical composition is provided in a single use container, including, for example, single-use vial, ampoule, syringe or autoinjector, whereas a multi-use container is provided in other embodiments. The vectors disclosed herein may be administered to a subject in any appropriate manner known in the art.

[0109] The vectors may be present in a "therapeutically effective amount", "effective amount" or "sufficient amount" which mean a quantity sufficient to achieve a desired result when administered to a subject. Therapeutically effective amounts may vary according to factors such as the age, sex, and weight of the subject. Dosage or treatment regimens may be adjusted to provide the optimum therapeutic response, as is understood by a skilled person.

[0110] In an embodiment, there is disclosed a composition of nucleic acid delivery vectors in a buffered solution for therapeutic administration.

[0111] Methods and Uses

[0112] The disclosed nucleic acid delivery vectors may be used to deliver a nucleic acid payload to a target cell. A target cell may be a mammalian cell, for example, a human cell. Once delivered to the target cell the encapsulated minivector may be internalized by the target cell. In some embodiments, the nucleic acid payload may then be expressed by the target cell machinery. The deoxyribonucleic acid may be used for any biological purpose that would benefit from the delivery of a deoxyribonucleic acid to a cell.

[0113] In an embodiment, the nucleic acid payload for delivery to a target cell comprises a DNA vaccine. The delivery of the nucleic acid payload may be useful for vaccination. In another example, the deoxyribonucleic acid may comprise an imaging agent or a diagnostic agent and may be used, for example, in the diagnosis of disease or in monitoring disease progression. The delivery of deoxyribonucleotides to a mammalian cell may be useful for the treatment or prevention of a disease or disorder. In an embodiment, the delivery of a nucleic acid payload to cells may be useful for tumor immunotherapy. In an embodiment, the nucleic acid payload may be used for gene transfer or gene editing techniques and the delivery of such deoxyribonucleic acids would be useful for treating or preventing a disease or disorder in a subject in need thereof. The disease or disorder to be treated encompasses any disease or disorder that would benefit from the delivery of a deoxyribonucleic acid to a cell.

[0114] In an embodiment, the nucleic acid delivery vector may be used to deliver a therapeutic or reporter transgene. In some embodiments the nucleic acid delivery vector may be used for immune-stealth gene delivery to hematopoietic, hepatic, pulmonary, ocular or mucosal tissues.

[0115] Anelloviruses have not been associated with any diseases in humans and are found in high abundance in various tissues and cell types. Vectors based on anelloviruses are non- immunogenic and may be redosable. In addition, anelloviruses transfect different cell types and tissues and travel to exosomes making them suitable and safer candidates for gene delivery. The inventors are not aware of any animal models or cell culture systems available for the propagation of anelloviruses. The methods disclosed herein overcome production limitations of producing cost-effective and safer viral vectors.

[0116] The vectors disclosed herein may be used as a gene therapy platform. The vectors may be used to deliver a payload nucleic acid (gene of interest) to a cell.

[0117] The vectors may be used, for example, for gene therapy. Gene therapy may be useful to treat and / or cure a myriad of diseases including, for example, cancer, immunodeficiency, and cardiovascular diseases. The basic concept of gene therapy involves the introduction of a gene into a target cell which, either leads to treating or the slowing down of the progression of the disease. Gene therapy may be carried out in the target cell ex vivo or in vivo. The in vivo method directly involves the administration of desired genetic material into the targeted cells or tissue.

[0118] In an embodiment, the nucleic acid delivery vector may be used for repeat-dose systemic or localized gene therapy without eliciting adaptive immune neutralization.

[0119] In an embodiment, there is provided a method of binding a targeting moiety to a target molecule of a target cell comprising contacting said target cell with the vectors as defined herein. In an embodiment there is provided a method of delivering a payload nucleic acid to a target cell comprising contacting said target cell with the vectors as defined herein.

[0120] In an embodiment, there is provided a use of the vector as defined herein for preparation of a composition for delivery of a nucleic acid payload to a cell.

[0121] In an embodiment, there is taught a scalable process to purify and isolate nucleic acid delivery vectors disclosed herein using rapid-sequential chromatography, wherein the isolated nucleic acid delivery vectors are high-titer and endotoxin free.[01221 Kits

[0123] The nucleic acid delivery vectors or the compositions disclosed herein may be packaged as a kit. There are also provided herein kits comprising a compound or compositions as described herein. Kits are generally in the form of a physical structure housing various components and may be used, for example, in practicing the methods provided herein (provided e.g. in a sterile container), which may in the form of a pharmaceutical composition suitable for administration to a subject.

[0124] The kit may also comprise diluents (e.g. sterile water), buffers, pharmaceutically acceptable excipients and the like. A kit may also contain a label or packaging insert including identification information for the components therein and instructions for their use. Labels or inserts can include manufacturer information such as lot numbers and expiration dates.

[0125] In an embodiment, the kit comprises: a DNA vector plasmid comprising: a viral DNA binding sequence; and a nucleic acid payload, wherein the viral DNA binding sequence and the nucleic acid payload are flanked by an M13 f1 origin of replication start sequence and an M13 f1 origin of replication STOP sequence; a helper plasmid for producing and packaging single stranded DNA containing the nucleic acid payload, the helper plasmid comprising all the genesrequired for M13 replication, transcription and packaging; and a plasmid for expressing ORF1 capsid proteins or portions thereof in prokaryotic cells. Where features are named herein, it will be understood that corresponding example sequences for the features (or sequences that comprise) may found in Table 2.EXAMPLE 1

[0126] This Example outlines the production of nucleic acid delivery vectors comprising capsid proteins or portions thereof which encapsidate DNA. The assembly of vectors was demonstrated using scanning electron microscopy, and real-time PCR.

[0127] TTV19 anellovirus capsid proteins were expressed and purified in E. coli BL21 cells. The ssDNA packaged by the anellovirus capsid proteins was produced using an M13 replication system in tandem via a miniphagemid processing technology. E. coli JM109 was used for DNA vector plasmid amplification and production of M13 phagemids that package ssDNA containing an anellovirus packaging signal. The plasmids used in the study are listed in Table 3.Table 3: Plasmids

[0128] The ORF1 sequence of TTV19 obtained from NCBI (GenBank: AB025946.2) was codon- optimized for protein production in bacteria using the Gensmart™ codon optimizing tool software. A 6X His-Tag (encoded by pET vector) was added to the N-terminal of TTV19 ORF1 along with a TEV cleavage, which allows for the removal of the His-tag if desired. The His-Tag is be used for protein detection and purification. The sequence was synthesized and added to the pET-28 vector. Figure 6 shows the pET-28_TTV19 Cap vector. The pET-28_TTV19 Cap vector consists of a T7 promoter, a lac operator, RBS, 6X His-Tag, TEV cleavage site, ORF1 of TTV19, and the kanamycin resistance gene.

[0129] The TTV19 intergenic region containing the DNA packaging sequence (GenBank: AB025946.2) was synthesized and introduced into the pCMV_gfp plasmid (Wong et al, 2023), and the construct was named pTTV19IR_CMV_gfp_M13 (Figure 7). Figure 7, Panel A depicts an M13 split origin of replication with a nucleic acid payload flanked between the M 13 start and M 13 stop sequences. Also flanked between the M 13 start and M13 stop sequence is an intergenic region comprising the DNA packaging sequence, also referred to as the DNA binding sequence. In Figure 7, Panel B depicts the pTTV19IR_CMV_gfp_M13 (left) and the plasmid after M13 replication and assembly into an M13 phagemid (i.e wherein the prokaryotic viral elements have been deleted), (right). The DNA vector plasmid consists of the M13 start sequence, TTV19 intergenic region, CM enhancer, CM promoter, gfp, SV40 poly (A) signal, and M13 stop sequence.

[0130] The helper plasmid pM13SW8 (Wong et al., 2023) was used to produce and package ssDNA containing the TTV19 intergenic region from the DNA vector plasmid. The helper plasmid comprises all the necessary genes from M13KO7 for M13 replication and transcription. The plasmid map of pM13SW8 is depicted in Figure 8.

[0131] Calcium-competent E. coli cells were transformed by vectors and plated on LB plates + selective antibiotic overnight and plates were observed for growth the next day. The transformed colonies were maintained on LB plates + antibiotic. Transformation of the cells was confirmed by plasmid extraction and restriction enzyme digestion with BamHI HF and AGE analysis. Verification of successful transformation of the plasmids and correct plasmid band sizes after restriction digestion was confirmed.

[0132] TTV19 capsid protein expression in E. coli BL21 was evaluated following IPTG induction at various optical densities. The expression of the protein was confirmed using SDS- PAGE and western blot analysis (Figure 9). In Figure 9 lane 1 is protein molecular weight ladder in kDa; lane 2 is IPTG induction at Aeoo = 0 which showed the presence of protein band at 75kDa; lane 3 is IPTG induction at Aeoo = 0.4 which showed the presence of protein band at 75kDa; lane 4 is IPTG induction at Aeoo = 0.6 which showed the presence of two protein bands at 75kDa and 25kDa; lane 5 is IPTG induction at Aeoo = 0.8 which showed the presence of two protein bands at 75kDa and 25kDa.

[0133] The expressed capsid proteins were purified using His60 Ni superflow resin and a gravity column kit. The targeted protein was eluted with approximately 10 column volumes of elution buffer and, 1 ml fractions were collected. The eluted samples were subjected to SDS- PAGE and western blot analysis, to detect the presence of the capsid proteins in the eluted samples (Figure 10 Panel A). The expected molecular weight of the TTV19 capsid protein was 75 kDa. Expressed proteins in crude extracts produced lighter protein bands at ~75 kDa, along with another protein band at ~50 kDa. Figure 10 Panel B shows TTV19 capsid protein from E. coli cell lysate detected by Western Blot. Lane 1 is the protein molecular weight ladder. Lanes 2-4 are crude extract samples. Lane 2 shows protein bands at 75 kDa and 25 kDa and Lanes 3-4 show protein bands at 75 kDa and 50 kDa, indicating that the TTV19 capsid proteins were present in the crude extracts.

[0134] Once the proteins were purified, proteins that were ~25 kDa in size were observed in elution fractions 1 , 2, and 3 following purification. Figure 11 Panels A-D show the SDS-PAGE and Western blot analysis of fractions during purification of His-tagged TTV19 capsid proteins using an anti-His antibody. The samples analyzed were crude extracts (CE) prior to purification, fractions collected before washing the columns upon sample addition (B) and fractions collected during elution (E). In Panel A, Lane 1 is protein molecular weight ladder, Lanes 2 and 3 are crude extracts containing TTV19 capsid protein and Lanes 4 and 5 are the first and second fractions (B1 and B2) collected by washing the columns before sample elution. Figure 11 Panel B shows B5, B4, and B3 respectively in Lanes 1 , 2, and 3 while Lane 4 is protein molecular weight ladder. Figure 11 Panel C shows a protein molecular weight ladder in lane 1 , protein fractions after elution, E1 , E2, E3, E4 and E5 in lanes 2-6, respectively. Figure 11 Panel D shows eluted proteins samples E5-E10 in lanes 1-6, respectively and Lane 7 is protein molecular weight ladder. Protein bands at 75 kDa and 50 kDa were found in the crude extracts, indicating the TTV19 capsid protein was present. Protein bands of 25 kDa were found in the E1 , E2 and E3 elution fractions. The TTV19 capsid protein appears to have been further processed during purification. The elutionfractions after Ni column purification only have the 25 kDa TTV19 capsid protein, also referred to as minimal capsid protein. The amount of protein present in the purified fractions was determined using a Pierce BCA Protein Assay KitPurified protein and the crude extract containing the capsid proteins were both used for further in vitro assembly of virus particles.

[0135] The concentration of purified protein samples was determined for in vitro virus assembly. A standard curve was obtained for albumin concentrations. From the standard graph, a linear equation was obtained which is y = O.OOOx + 0.0211 and R2value of 0.9986. Using this formula, the unknown concentration of purified TTV19 capsid proteins was determined. From all the purification steps carried out during the study, the average concentration of the protein sample was found to be 39.4985 ± 9.148 pg / ml. The purified TTV19 capsid protein samples were used for in vitro assembly of ssDNA.

[0136] Production of ssDNA containing the TTV19 DNA binding sequence and qfp using an M13 replication system

[0137] Synthetically originated ssDNA was packaged by TTV19 capsid protein in vitro. This was achieved by using the M 13 phage replication system using the helper plasmid, M13SW8, and the DNA vector plasmid, pTTV19 IR_CMV_gfp_M13, and produced in vivo in E. coli host cells. The helper plasmid encodes the proteins necessary for M13 phage replication, transcription and assembly of M13 phagemids intracellularly in bacterial systems (Wong et al., 2023). The M13 genome consists of an origin region that has nucleotide sequences for initiation and termination of replication. While the ssDNA to be packaged by M13 phagemids was produced using a DNA vector plasmid, the M13 origin of replication was separated and flanked by a mammalian transgene expression cassette, isolating it from the bacterial backbone (Wong et al, 2023). This permits the separation of the M13 initiation sequence from the termination sequence and the ability to clone a nucleic acid payload between the initiation and termination gene sequences without any intervening prokaryotic sequences or helper phage contamination (Wong et al, 2023). This approach is disclosed in PCT / CA2023 / 050586. Using this miniphagemid approach, M13 phagemids were produced that packaged a ssDNA payload and the TTV19 DNA binding signal.

[0138] E. coli JM109 cells were transformed with pTTV19 IR_CMV_gfp_M13 (for packaging of ssDNA) and the helper plasmid (pM13SW8) for M13 phagemid production. A plaque assay was conducted to determine phage concentration. The average phage concentration was found to be 1.53 x 109± 1.66x 108phage / ml. Calibration curves were used to estimate phage concentration. The calibration curve for gfp gene region was measured from qPCR of pTTV19 IR_CMV_gfp_M13 plasmid.

[0139] After M13 phagemids were produced and purified, ssDNA was extracted from M13 phagemids. The extracted ssDNA was visualized using AGE and appeared on the gel at the expected size of ~ 0.8 kb.

[0140] The M13 phagemid lysate was filtered through a 60 ml syringe using a 0.22 pm filter into a fresh 50 ml centrifuge tube and subjected to polyethylene glycol (PEG) precipitation. The filtered lysate was concentrated by adding 1 / 5ththe volume in PEG / NaCI and mixed by inversion. The mixture was incubated at 4°C for 2 h and centrifuged at 12,000 X g for 15 min at 4°C. The white pellet was re-suspended in cold TN buffer and a second PEG precipitation was carried out to further concentrate the phagemid. PEG-precipitated phagemid was then treated with DNasel for 1 h at 37°C to remove any contaminating bacterial DNA. The concentrated phagemid was stored at 4°C.

[0141] The PEG-concentrated phagemid lysate was mixed with phenol (1 :1 , v / v) and subsequently vortexed. The mixture was centrifuged at 4° C for 5 min, and the top aqueous layer was extracted. This layer was extracted again with an equivalent volume of phenol: chloroform twice and once with chloroform. The phagemid ssDNA was precipitated overnight in 95% ethanol at -80° C. The precipitated ssDNA was washed twice with 70% ethanol, dried, and re-suspended in DNase and RNase- free water. The ssDNA was quantified using Nanodrop analysis. The ssDNA was then subjected to gel electrophoresis to confirm the size of the ssDNA.

[0142] A plaque assay was carried out to quantify viable M13 phagemid. E. coli strain JM109 was used for carrying out plaque assay as it is an F+strain suitable to produce filamentous phages. A series of M 13 phagemid dilutions were prepared in TN buffer to obtain countable (30-300) plaques on a spot plate. 200 pL of early log-phase of E. coli strain JM109 was mixed with 3 ml of 0.8% top agar supplemented with 5 mM MgSO4, which was then poured on a pre-warmed LB agar plate. A 5 pL sample of each serially diluted M13 phagemid was then spotted on the top agar. The plates were then incubated at 37°C for 4-8 h. The lowest dilution that generated distinct plaques on the bacterial lawn was selected to generate full plates for plaque assay. The full plate plaque assay was carried out using the same procedure, instead of spotting the phagemids on the top agar, the phagemids were mixed directly into the bacterial culture before adding to the top agar and pouring it on the LB plates. The titre was determined from the mean of three counts and expressed as plaque-forming units (PFU) per millilitre (ml).

[0143] In vitro assembly of vectors

[0144] In vitro assembly of capsid proteins and the extracted ssDNA encapsidated in TTV19 capsid protein was achieved by assembling His-Tag purified TTV19 capsid protein and ssDNA.Along with His-Tag purified capsid proteins, crude extract containing TTV19 capsid protein and ssDNA were also assembled in vitro.

[0145] Capsid proteins were suspended in 5X assembly buffer (pH 7.2, 250 mM Tris- HCI buffer containing 250 mM NaCI, 50 mM KCI, and 25 mM MgCh) at a final concentration of 7.5 mg / ml. A 1X assembly buffer solution containing 50 mM Tris-HCI, 50 mM NaCI, 10 mM KCI, and 5 mM MgCh with a pH of 7.2 was created by combining capsid protein and ssDNA in a 6:1 mass ratio (w / w) of capsid protein to ssDNA and a 4:1 (v / v) ratio of Milli-Q water to buffer. The capsid proteins and ssDNA were incubated at 4°C overnight before analysis and purification. Capsid proteins suspended in an assembly buffer without ssDNA was used as the negative control for qPCR and functional assembly. Similarly, ssDNA in the absence of protein served as an unencapsidated control for DNase digestion assays.

[0146] Anellovirus capsid-assembled ssDNA and protein and / or ssDNA only control was subjected to DNase treatment. 1 pL of DNase enzyme was added to the samples. The samples were then incubated at 37° C for 1 h. After the treatment of samples with DNase, the samples were treated with 1 L of DNase stop solution and incubated at 65°C for 15 min. To visualize ssDNA digestion AGE was performed. The samples were stored at 4°C.

[0147] lodixanol ultracentrifugation.

[0148] The crude protein extract and assembled ssDNA with the crude capsid protein were subjected to iodixanol ultracentrifugation. The iodixanol gradient (10-60%) was prepared using OptiPrep™ and 1X NTC solution. To prepare a step gradient, a 1 .5 ml layer of each concentration starting from the highest to the lowest (60% — > 10%) was added to thin wall tubes for the SW41 rotor (polyallomer centrifuge tubes, 14 X 89 mm; Beckman Coulter, 331372). The gradient tube was then stored at 4° C. Alternately, the samples were prepared by pelleting through ultracentrifugation in an SW41 T1 rotor at 4° C for 90 min at 26,000 rpm. The pellet was resuspended in chilled 1X NTC solution. The samples were then layered onto the iodixanol density gradient, followed by ultracentrifugation in an SW41 T1 rotor at 4° C for 150 min at 35,000 rpm. After ultracentrifugation, 12 fractions (with a volume of ~ 1 mL each) were collected for each sample by puncturing a hole within the centrifuge tubes from the top to the bottom of the tube.

[0149] The purified samples were then subjected to SDS-PAGE and western blot to detect the presence of purified capsid protein in the fraction. The purified proteins for both samples were obtained in fraction 10 at a band size of ~ 25 kDa. Fraction 10 of the iodixanol centrifuged samples of crude extract containing TTV19 capsid protein, and fraction 10 of the iodixanol centrifugedsamples of assembled ssDNA and crude extraction containing TTV19 capsid protein were used for further study.

[0150] Scanning Electron Microscopy (SEM).

[0151] The assembled capsid proteins and ssDNA (nucleic acid delivery vectors) were visualized using SEM, STEM and high-resolution TEM. TTV19 virus is ~ 50nm and assembles as an icosahedral head. Figure 12 shows SEM images forTTV19 anellomids produced in vivo or in vitro according to certain embodiments of the invention. Figure 12, Panel A shows SEM images of in vivo assembled anellomids, produced using the one-step method further described in Example 3. Figure 12, Panel B and Panel C show SEM images of in vitro assembled anellomids. SEM was performed using Zeiss Merlin High-resolution SEM and ZeissSmartSEM software. The Zeiss Merlin High-resolution SEM contains a GEMINI II column, which is the area of the Field Emission SEM, where electrons are emitted, accelerated, bundled, focused and deflected. Vector samples are loaded into the SEM, placed under vacuum and images were captured. Figure 13 shows scanning transmission electron microscopy (STEM) images of in vitro assembled anellomids. Figure 14 shows a high resolution transmission electron microscopy (TEM) image of in vivo assembled anellomids, produced according to the one-step procedure further explained in Example 3. TEM and STEM images were captured with a Hitachi HT7830 TEM microscope. Prepared samples were added to carbon-coated copper TEM grids. The TEM grids were loaded into the TEM microscope and TEM or STEM images were captured.

[0152] Quantitative PCR (qPCR)

[0153] ssDNA packaging by capsid proteins was confirmed by quantitative PCR (qPCR). qPCR was also used to determine the concentration of assembled virus particles in vitro. To estimate virus concentration, the standard calibration curve was generated using the plasmid pTTV19 IR_CMV_gfp_M13. A calibration curve for quantifying virion particles was constructed using the pTTV19 IR_CMV_gfp_M13 plasmid that encodes the gfp gene. The gfp gene region was used for the amplification of the DNA samples, where 100-fold serial dilutions (gc / pl) of the plasmid were made (10° -108) to generate the calibration curve.

[0154] For each qPCR amplification, the PCR reaction was prepared using 5 pL of Powerllp SYBR Green Mix, 1 pL each of 500 nM primer (forward and reverse), 2 pL of the DNA sample, and 1 pL of dH2O for a final reaction volume of 10pL. PCR cycling conditions were as follows: 50° C for 2 min, 95° C for 2 min, followed by 40 cycles at 95° C for 15 s and 60° C for 1 min. Next, the melt curve was set for 1 cycle at 95° C for 15 s, 60° C for 1 min, and 95° C for 15 s. PCR reactionswere run in triplicate on the StepOne Plus Real-Time PCR system (Applied Biosystems, Waltham, USA). The quantification cycle or threshold cycle number (Ct) for each reaction was recorded and used in subsequent analysis.

[0155] The following equation converts the mass of dsDNA to the quantity of genome copies:Concentration of template DN AX6.022X1023gc = ■where gc is the concentration of virion genome Length of the DNA (bp)xlx!09x660 copies (genome copies / pL). Virus concentrations were estimated from their respective calibration curve. First, the Ct values from the control were plotted against the log of the known concentrations for each concentration in the dilution series. Linear regression produces a familiar equation: Ct = mx + b where Ct is the measured threshold cycle number, m is the slope, x is the base-10 log of the concentration in gc / pL and b is the x-intercept. PCR amplification efficiency is given by E = 10"1 / m- 1. The virion concentration, V can be estimated simply by the equation: V = 10(ct-b) / mx2 where multiplication by 2 adjusts for the estimation of single-stranded products (gc / pL) from dsDNA standards. Only Ct measurements within the bounds of the calibration curve were used to calculate virion concentration.

[0156] qPCR analysis of the samples showed that the ssDNA that was generated by M13 processing was successfully packaged by crude extract containing TTV19 capsid proteins as well as His-Tag purified TTV19 capsid proteins based on the amplification curves obtained as well as threshold cycle number (Table 4). ssDNA extracted using heat treatment as well as by phenolchloroform from assembled capsid protein from the crude extract and from assembled His-Tag- purified capsid proteins was successfully amplified, indicating that the DNA was packaged by the capsid proteins. As expected, ssDNA treated with DNase enzyme did not show any amplification. Using the threshold cycle number, the concentration of virus particles was determined as shown in Table 4.Table 4: Ct Values and virion concentration for the qPCR amplified samples

[0157] The concentration of virus particles produced in the study was similar for both ssDNA assembled by crude extract containing TTV19 capsid protein and His-tag purified TTV19 capsid protein. The vectors were assembled ex vivo in a cell extract system when combined, which was demonstrated using scanning electron microscopy, and real-time PCR. Purified TTV19 capsid proteins and crude extract containing TTV19 capsid proteins were both able to encapsidate ssDNA within the capsid proteins and self-assemble into nucleic acid-containing vectors. His-tag purified proteins, which were 25 kDa in size, were able to assemble ssDNA.

[0158] Production of TTV19 anellomid delivery vectors with luciferase nucleic acid payload

[0159] TTV19 nucleic acid delivery vectors, also referred to as anellomids, containing a luciferase gene expression cassette (TTV19-luciferase vectors) were produced using the method described above for TTV19 delivery vectors. Figure 15 depicts the plasmid map for pRP5 containing an M13 split origin of replication with a TTV19 intergenic region and a nucleic acid payload, namelya luciferase gene expression cassette flanked between the M 13 start and M13 stop sequences. Figure 16 shows concentration data determined by qPCR for the luciferase anellomid and the gfp anellomid. The assembled TTV19 delivery vector concentration was increased by 10000-fold for the luciferase anellomid (4.26x108) compared to the gfp anellomid (4.19x104). Therefore, the anellomid concentration was increased by 10,000-fold when the size of payload DNA was increased from 1 ,6kb to 3.8kb, which is equivalent to the size of the TTV19 genome

[0160] After in vitro assembly of nucleic acid delivery vectors, the concentration of His purified TTV19 capsid proteins encapsidating ssDNA (luciferase) was determined using qPCR and Dynamic Light Scattering (DLS). DLS also showed vector size and polydispersity. The results of the qPCR and DLS analysis for TTV19-luciferase vectors is shown in Table 5.Table 5. Concentration, vector size and polydispersity of TTV19-luciferase vectors

[0161] The capacity of TTV19-luciferase vectors to transfect mammalian cells was determined using the human embryonic kidney (HEK) 293T cell line. HEK 293T cells were transfected with the assembled TTV19 anellovirus vectors carrying a luciferase gene expression cassette and compared against dsDNA and ssDNA carrying the GOI at a multiplicity of infection (MOI) of 20. Luminescence was measured after 96 hours. Figure 17 shows the luminescence detected in TTV19-luciferase (anellomids) transfected cells with or without Turbofect™ transfection reagent, dsDNA and ssDNA luciferase transfected cells with Turbofect™ reagent (positive controls) as well as the negative control. Transfection was detected in all samples except the negative control. TTV19-luciferase vectors transfected HEK 293T cells with or without Turbofect™ reagent.EXAMPLE 2

[0162] This example outlines a protocol for the in vitro assembly of TTV19 anellosomes for gene therapy. BL21 (DE3) E. coli cells were transformed with the pET-28_TTV19 Cap plasmid encoding His-tagged TTV19 capsid protein. The transformed cells were grown in LB broth+ Kanamycin (Kn)until the culture reached an optical density (OD) of Aeoo = 0.6 at 37°C. Once the OD reaches 0.6, protein expression was induced by adding 1 mM IPTG to the culture. The cells were grown for an additional 4-5 hours to maximize protein production. The cells were pelleted by centrifuging at 10,000 x g for 10 minutes at 4 °C. The cell pellet was washed twice with cold Phosphate-buffered saline (PBS). The cell pellet was resuspended in NEBExpress E. coli Lysis Reagent by pipetting or vortexing briefly until the suspension was homogenous. 0.025 - 0.075 mL of NEBExpress E. coli Lysis Reagent was used for every 1 UODeoo harvested. To calculate the UODeoo, the volume harvested is multiplied by the ODeoo reading. For example, a 5 mL culture harvested at ODeoo 1 gives 5 mL x 1.0 = 5 UODeoo. In this example, 0.125 - 0.375 mL Lysis Reagent is required to lyse efficiently. If harvested cells are weighed, 5 mL of NEBExpress E. coli Lysis Reagent is used per1 gram of cells. The resuspended cells were incubated at room temperature for 10 - 20 min with gentle shaking, gentle rotation, or swirling. Lysis is usually visible with a clearance of the suspension. The lysate was centrifuged at 16,000 x g for 10 min at 4°C to pellet the insoluble material and cell debris (30 min or longer for large volumes and lower speed). The supernatant was carefully transferred into a sterile container for analysis or purification. This soluble fraction can be stored at 4°C for a few hours or -20°C or -80°C for longer term storage.

[0163] The extracted protein was purified using Ni-NTA column purification. The Ni-NTA agarose resin and associated buffers were equilibrated to 4°C. The top and bottom stoppers of the column were removed and the column was washed with 5 column volumes of equilibration buffer and 5 column volumes of NEBExpress E. coli Lysis Reagent. The equilibration buffer has 50 mM sodium phosphate, 8 M urea, 300 mM NaCI, 20 mM imidazole at pH 7.4. The extracted protein sample was added to the equilibrated column and the column was inverted slowly, incubating at 4°C for2 hours to allow the protein to bind. The column was installed in a vertical position and the resin was allowed to settle. The column was washed with 10 column volumes of equilibration buffer followed by 10 column volumes of wash buffer. The wash buffer has 50 mM sodium phosphate, 8 M urea, 300 mM NaCI, 40 mM imidazole at pH 7.4. The target protein was eluted by adding 10 column volumes of His60 Ni elution buffer and 1 mL fractions were collected. The elution buffer has 50 mM sodium phosphate, 8 M urea, 300 mM NaCI, 300 mM imidazole at pH 7.4. The eluted fractions were subjected to SDS-PAGE and Western blot to confirm the presence of the His- tagged protein.

[0164] To produce ssDNA for in vitro assembly of anellosomes, a fresh colony of calcium competent E. coli JM109 containing plasmids M13SW8 and TTV19 IR_CMV_gfp_M13 or TTV19 IR_CMV_luciferase_M13 (pRP5) was grown in LB broth + Kanamycin (Kn) + 5mM MgSO4 until the culture reaches an optical density (OD) of Aeoo = 1 .0 at 37°C. The bacterial culture wascentrifuged at 8,000 x g for 10 minutes to separate the bacterial pellet from the supernatant containing the phage lysate. The M13 miniphagemid lysate was filtered using a 0.22 pm filter through a 60 mL syringe into a fresh 50 mL centrifuge tube. The filtered lysate was concentrated by adding 1 / 5ththe volume of PEG / NaCI solution, mixing gently by inversion and incubating at 4°C overnight. The mixture was centrifuged at 12,000 x g for 15 minutes at 4°C. The supernatant was discarded and the pellet was centrifuged again at 12,000 x g for 5 minutes at 4°C. The pellet was re-suspended in 1 ml cold TN buffer to further concentrate the phagemid. A second PEG precipitation was performed adding 1 / 5ththe volume of PEG / NaCI solution, mixing gently by inversion and incubating at 4°C overnight. The mixture was centrifuged at 16,000 x g for 15 minutes at 4°C. The supernatant was discarded and the pellet was centrifuged again at 16,000 x g for 5 minutes at 4°C. The pellet was re-suspended in 10OpI cold TN buffer. The concentrated phagemid was treated with DNase I for 1 hour at 37°C to remove any contaminating bacterial DNA. The miniphagemid was then treated with DNase Stop solution for 15 mins, at 65°C. The final concentrated miniphagemid was stored at 4°C for further use. The miniphagemid was quantified using qPCR.

[0165] The PEG-concentrated phagemid lysate was mixed with phenol (1 :1 , v / v) and vortexed thoroughly. The mixture was centrifuged at 4°C for 5 minutes and the top aqueous layer collected. The aqueous layer was extracted twice with an equal volume of phenokchloroform, followed by one extraction with chloroform. The ssDNA was precipitated by adding 95% ethanol and incubating the mixture overnight at -80°C. The precipitated ssDNA was washed twice with 70% ethanol, air-dried, and re-suspended in DNase and RNase-free water. The ssDNA was quantified using Nanodrop spectrophotometry. To confirm the size of the ssDNA, the sample was subjected to gel electrophoresis.

[0166] TTV19 anellosomes are assembled in vitro by combining the His-tag purified TTV19 capsid protein and the extracted ssDNA from miniphagemid. The capsid proteins were suspended in 5X assembly buffer to a final concentration of 7.5 mg / ml. The 5X assembly buffer contains 250 mM Tris-HCI (pH 7.2), 250 mM NaCI, 50 mM KCI and 25 mM MgCI2. The capsid protein was mixed with ssDNA in a 6:1 mass ratio (w / w) of capsid protein to ssDNA. A 4:1 (v / v) ratio of Milli-Q water to 1X assembly buffer was added to the protein-DNA mixture. The mixture was incubated at 4°C for at least overnight to allow the in vitro assembly of the viral particles. A Negative Control was prepared from a solution containing capsid proteins in the assembly buffer without adding ssDNA. This negative control can be used for qPCR and functional assembly analysis. An unencapsidated control was prepared from a solution containing ssDNA without capsid proteins.This unencapsidated control can be used for DNase digestion assays. After overnight incubation, encapsidated ssDNA was quantified using qPCR.EXAMPLE 3

[0167] This example outlines the production and complete synthetic assembly of anelloviruses in vivo in an E. coli system. This is also described herein as a “one step” procedure. Figure 18 depicts an embodiment of the one step method wherein a helper plasmid generates M13 phage proteins along with TTV19 capsid protein in E. coli. In the same E.coli cell, a DNA vector plasmid generates ssDNA with a DNA packaging sequence and nucleic acid payload. The ssDNA is packaged by the TTV19 capsid proteins generating the anellosomes vectors. The assembled vectors may be obtained by lysing the cells. A “one step” method can be achieved by replacing M13 major coat protein, gpVIll with anellovirus ORF1 in helper plasmid M13SW8, which helps with the expression of capsid proteins as well as the assembly of ssDNA intracellularly.

[0168] The p8 gene from the M13SW8 plasmid was deleted and the nucleic acid encoding the TTV19 capsid protein sequence was obtained from pET-28_TTV19 Cap and cloned into M13SW8 in place of p8 to form pRP2_75kDa. The plasmid map of pRP2_75kDa is shown in Figure 19 and the plasmid sequence is outlined in Table 2. It was observed that deleting the gV gene from pRP2_75kDa increased the concentration of anellomids produced. The plasmid is pRP2_75kDa_gV deletion and the plasmid map is shown in Figure 20.

[0169] To assemble ssDNATTV19 anellosomes in vivo, a fresh colony of E. coli JM 109 containing plasmids pRP2_75kDa and TTV19IR_CMV_gfp_M13 or TTV19IR_CMV_luciferase_M13 (pRP5) was grown in LB + Kanamycin (Kn) + 5mM MgSCL broth until the culture reached an optical density (OD) of Aeoo = 1.0 - 1.2 at 37°C. The bacterial culture was centrifuged at 8,000 x g for 10 minutes at 4°C. The cell pellet was washed twice with cold PBS. The cell pellet was resuspended in NEBExpress E. coli Lysis Reagent by pipetting or vortexing briefly until the suspension is homogenous. 0.025 - 0.075 mL of NEBExpress E. coli Lysis Reagent was used for every 1 UODeoo harvested. To calculate the UODeoo, multiply the volume harvested by the ODeoo reading. For example, a 5 mL culture harvested at ODeoo 1 gives 5 mL x 1 .0 = 5 UODeoo. In this example, 0.125 - 0.375 mL Lysis Reagent is required to lyse efficiently. If harvested cells are weighed, use 5 mL of NEBExpress E. coli Lysis Reagent per 1 gram of cells. The resuspended cells are incubated at room temperature for 20 min with gentle shaking, gentle rotation, or swirling. Lysis is usually visible with a clearance of the suspension. The lysate is centrifuged at 12,000 x g for 15 min at 4°C to pellet the insoluble material and cell debris (30 min or longer for large volumes and lower speed). The supernatant is carefully transferred into a sterile container for purification.The lysate was filtered using a 0.22 m filter through a 10 ml syringe into a fresh 15ml centrifuge tube. The filtered lysate was concentrated by adding 1 / 5ththe volume of PEG / NaCI solution, mixing gently by inversion and incubating at 4°C overnight. The mixture was centrifuged at 12,000 x g for 15 minutes at 4°C. The supernatant was discarded and the pellet centrifuged again at 12,000 x g for 5 minutes at 4°C. The pellet was re-suspended in 1 mL cold TN buffer to further concentrate the phagemid. The concentrated anellosomes were treated with DNase I for 1 hour at 37°C to remove any contaminating bacterial DNA followed by treatment with DNase I Stop solution for 15 mins at 65°C. The final concentrated anellosomes were stored at 4°C for further use. The anellosomes were quantified using qPCR.

[0170] E. Coli cells were transformed with pRP2_75 kDa_gV deletion and pRP5 for production of nucleic acid vectors in vivo. The vectors were purified from the E. coli lysate and tested for the encapsidation of DNA. The concentration of in vivo assembled vectors, also referred to as anellomids, was determined by qPCR. Deleting the gV gene from pRP2_75kDa induced a significant boot in the concentration. The qPCR protocol from Example 1 , in vitro production of vectors, was used with primers for the luciferase gene of interest. Figure 21 shows that the concentration of in vivo assembled anellomids after purification by PEG concentration or sephacryl column and in vitro assembled anellomids. The concentration of in vivo assembled anellomids determined by qPCR and DLS as well as vector size and polydispersity are shown in Table 6. The in vitro anellomids in Figure 21 were made using the plasmids pET-28_TTV19 Cap and pRP5, while the in vivo anellomids were made using pRP2_75 kDa_gV deletion and pRP5 plasmids. The results showed a significant increase in anellomid concentration achieved through in vivo assembly and subsequent purification methods compared to in vitro assembled anellomids. Both purification methods of the in vivo system (3.70E+11 and 7.43E+10) represent improvement over the in vitro assembly concentration (4.26E+08), suggesting in vivo assembly is an efficient strategy for obtaining high concentrations of purified anellomids. qPCR was also used to detect any helper plasmid contamination in the in vivo assembled anellomids. Figure 22 shows that no helper or DNA vector plasmid contamination was found in the anellomids. Therefore, the in vivo assembled anellomids package only the gene cassette of interest. Figure 23 shows the amplification plot and melt curve for qPCR experiments run to detect helper plasmid contamination. No helper backbone was detected in the in vivo assembled anellomids. The anellomids in Figures 22 and 23 were made using pRP2_75 kDa_gV deletion and pRP5 plasmids.Table 6. Concentration, vector size and polydispersity of TTV19-luciferase vectors

[0171] Figures 24 and 25 show the results of SDS-Page and Western blot detection of the TTV19-luciferase vector (anellomid) assembled in vivo. The membrane was blocked with BSA in TBS-T 7% (m / v), 10 mL prior to incubation with an anti-His primary antibody at 20 microliter in 10 mL of the blocking buffer. The secondary antibody used was an anti-mouse HRP conjugate. Histidine tagged TTV19-luciferase vectors assembled in vivo could be detected following PEG and SEC purification.

[0172] In Figure 24, following the first two molecular weight ladder lanes, lane 1 is an old TTV19 crude lysate, lane 2 is a new TTV19 crude lysate, lane 3 is TTV19 Ni-His purified, lane 4 is anellomid in vitro assembled old, lane 5 is anellomid in vivo assembled, PEG purified old, lane 6 is anellomid in vivo assembled, PEG purified new, lane 7 is anellomid in vivo assembled SEC purified old and lane 8 is ssDNA. The TTV19-luciferase vector assembled in vivo and purified by PEG showed protein bands at 70 kDa, 35 kDa and 25 kDa. The TTV19-luciferase vector assembled in vivo and purified by SEC showed protein bands at 35 kDa and 25 kDa. The TTV19 crude lysate showed protein bands at 100 kDa, 70 kDa, 40 kDa and 35 kDa.

[0173] In Figure 25 following the molecular weight ladder lane, lane 1 is ssDNA, lane 2 is anellomid in vivo assembled SEC purified old, lane 3 is anellomid in vivo assembled PEG purified new, lane 4 is anellomid in vivo assembled PEG purified old, lane 5 is anellomid in vitro assembled od, lane 6 is TTV19 Ni-His purified, lane 7 is TTV19 crude lysate new and lane 8 is TTV19 crude lysate old. The SEC purified TTV19-luciferase vector assembled in vivo showed a protein band at 35 kDa. The PEG purified TTV19-luciferase vector assembled in vivo showed protein bands at 70 kDa, 50 kDa, 35 kDa and 25 kDa. The TTV19 crude lysate in lane 7 showed protein bands at 100 kDa, 70 kDa, 40 kDa, 35 kDa and 25 kDa.REFERENCES

[0174] All references are hereby incorporated by reference herein, in their entirety.Bendinelli M, Pistello M, Maggi F, Fornai C, Freer G, et al. (2001) Molecular properties, biology, and clinical implications of TT virus, a recently identified widespread infectious agent of humans. Clin Microbiol Rev 14: 98-113.Bostan et al. (2013) Current and Future Prospects of Torque Teno Virus. Journal of Vaccines & Vaccination 2013 / 01 / 01. ISSN: 10.4172 / 2157-7560. S 1-004.Wong S., 2022. Construction and characterization of a hybrid phage gene delivery platform. (Doctoral Thesis). University of Waterloo, Waterloo, Canada.Wong, S., Jimenez, S., and Slavcev, R. 2023, Microb Cell Fact, 22(1):124.

Claims

WHAT IS CLAIMED IS:1 . A nucleic acid delivery vector comprising capsid proteins or a portion thereof from a nonenveloped mammalian virus, wherein the capsid proteins or portions thereof encapsidate a nucleic acid payload and wherein the capsid proteins or portions thereof were expressed and functionally processed in prokaryotic cells.

2. The nucleic acid delivery vector according to claim 1 , wherein the prokaryotic cells are E. coli cells.

3. The nucleic acid delivery vector according to claim 1 or 2, wherein the nucleic acid payload is a single-stranded deoxyribonucleic acid (ssDNA), a double stranded deoxyribonucleic acid (dsDNA), or a ribonucleic acid (RNA).

4. The nucleic acid delivery vector according to any of claims 1 to 3, wherein the nucleic acid payload is self-complementary and is a linear, covalently closed DNA.

5. The nucleic acid delivery vector according to any of claims 1 to 2, wherein the nucleic acid payload is circular.

6. The nucleic acid delivery vector according to any of claims 1 to 5, wherein the nucleic acid payload is a synthetic nucleic acid.

7. The nucleic acid delivery vector according to any of claims 1 to 6, wherein the nucleic acid payload is produced in eukaryotic cells, and wherein the eukaryotic cells are yeast or mammalian cells.

8. The nucleic acid delivery vector according to any of claims 1 to 7, wherein the nucleic acid payload is produced in prokaryotic cells.

9. The nucleic acid delivery vector according to any of claims 1 to 5 or 8, wherein the nucleic acid payload comprises filamentous phage-processed DNA.

10. The nucleic acid delivery vector according to any of claims 1 to 5 or 8, wherein the nucleic acid payload comprises M13-processed single stranded circular DNA.11 . The nucleic acid delivery vector according to any of claims 1 to 5 and 8 to 10, wherein the nucleic acid payload comprises a DNA minivector comprising a nucleic acid payload and afilamentous phage origin of replication, wherein the DNA minivector is devoid of prokaryotic vector backbone genetic sequences.

12. The nucleic acid delivery vector according to claim 11 , wherein the filamentous phage origin of replication is an M 13 origin of replication or an f1 origin of replication.

13. The nucleic acid delivery vector according to any of claims 1 to 12, wherein the nonenveloped mammalian virus is an anellovirus.

14. The nucleic acid delivery vector according to claim 13, wherein the anellovirus is from the genera Alphatorquevirus, Betatorquevirus, Deltatorquevirus, Epsilontorquevirus, Etatorquevirus, Gammatorquevirus, lotatorquevirus, Kappatorquevirus, Lambdatorquevirus, Mutorquevirus, Nutorquevirus, Thetatorquevirus, Zetatorquevirus, Gyrovirus, Chitorquevirus, Omegatorquevirus, Omicrontorquevirus, Pitorquevirus, Psitorquevirus, Rhotorquevirus, Sigmatorquevirus, Upsilontorquevirus, Xitorquevirus, Aleptorquevirus, Dalettorquevirus, Gimeltorquevirus, Hetorquevirus, Tettorquevirus, Wawtorquevirus or Zayintorquevirus.

15. The nucleic acid delivery vector according to any of claims 1 to 14, wherein the capsid protein or portion thereof comprises an anellovirus ORF1 capsid protein or an N-terminal portion of an anellovirus ORF1 capsid protein.

16. The nucleic acid delivery vector according to any of claims 1 to 14, wherein the capsid protein or portion thereof comprises the N-terminal 3rd portion of a TTV19 ORF1 capsid protein having a molecular weight of 25 kDa.

17. The nucleic acid delivery vector according to any of claims 1 to 15, wherein the capsid protein or portion thereof is encoded by a nucleic acid sequence comprising: the sequence set forth in SEQ ID NO: 1 ; SEQ ID: NO:2; or SEQ ID NO:3; a sequence having at least 80% sequence identity with the sequence set forth in SEQ ID NO:1 ; SEQ ID NO:2; or SEQ ID NO:3; a sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO:1 ; SEQ ID NO:2; or SEQ ID NO:3; a sequence having at least 95% sequence identity with the sequence set forth in SEQ ID NO:1 ; SEQ ID NO:2; or SEQ ID NO:3; or a sequence having at least 99% sequence identity with the sequence set forth in SEQ ID NO:1 ; SEQ ID NO:2; or SEQ ID NO:3.

18. The nucleic acid delivery vector according to claim 17, wherein the nucleic acid sequence is codon optimized for E. coli expression19. The nucleic acid delivery vector according to claim 17, wherein the nucleic acid sequence is CpG dinucleotide-depleted.

20. The nucleic acid delivery vector according to any of claims 1 to 15 or 17 to 19, wherein the capsid proteins or portions thereof comprise capsid proteins or portions thereof comprising: an amino acid sequence as set forth in SEQ ID NO:4 or SEQ ID NO:5; an amino acid sequence at least 80% identical to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5; an amino acid sequence at least 90% identical to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5; an amino acid sequence at least 95% identical to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5; or an amino acid sequence at least 99% identical to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5; wherein the amino acid sequence at least 80% identical, 90% identical, 95% identical or 99% identical to the sequence set forth in SEQ I D NO:4 or SEQ I D NO:5 retains the ability to selfassemble into the nucleic acid delivery vector.

21. The nucleic acid delivery vector according to any of claims 1 to 15 or 17 to 19, wherein the capsid proteins of portions thereof comprise capsid proteins or portions thereof comprising: an amino acid sequence as set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19; an amino acid sequence at least 80% identical to the sequence set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19; an amino acid sequence at least 90% identical to the sequence set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19;an amino acid sequence at least 95% identical to the sequence set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19; or an amino acid sequence at least 99% identical to the sequence set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19, wherein the amino acid sequence at least 80% identical, at least 90% identical, at least 95% identical or at least 99% identical to the sequence set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19, retains the ability to self- assemble into the nucleic acid delivery vector.

22. The nucleic acid delivery vector according to any of claims 1 to 21 , wherein the capsid proteins or portions thereof comprise an N-terminal tag.

23. The nucleic acid delivery vector according to claim 22, wherein the N-terminal tag is for purifying the capsid proteins or portions thereof and / or for purifying the nucleic acid delivery vector and / or for targeting the nucleic acid delivery vector to a target cell.

24. The nucleic acid delivery vector according to claim 22 or 23, wherein the N-terminal tag is a polyhistidine-tag, a FLAG-tag, NE-tag, Strep-tag, Spot-tag, T7-tag, or a TrpE-tag.

25. The nucleic acid delivery vector according to claim 24, wherein the tag is a polyhistidine tag (His-tag).

26. The nucleic acid delivery vector according to any of claims 1 to 25, wherein the capsid proteins or portions thereof were purified from E. coli cells.

27. The nucleic acid delivery vector according to claim 26, wherein the capsid proteins were purified by affinity chromatography.

28. The nucleic acid delivery vector according to claim 26, wherein the capsid proteins or portions thereof are N-terminally His-tagged and are purified by Ni column purification.

29. The nucleic acid delivery vector according to any of claims 1 to 28, wherein the prokaryotic cells are protease deficient.

30. The nucleic acid delivery vector according to claim 29, wherein the prokaryotic cells are BL21 E. coli cells.

31. A nucleic acid delivery vector comprising capsid proteins or portions thereof from a nonenveloped mammalian virus, wherein the capsid proteins or portions thereof encapsidate a nucleic acid payload and wherein the capsid proteins or portions thereof comprise an N-terminal portion of the TTV19 ORF1 capsid protein that has a molecular weight of approximately 25 kDa.

32. A nucleic acid delivery vector comprising TTV19 capsid protein or portions thereof, wherein the TTV19 capsid protein or portions thereof encapsidate a synthetic or modified circular ssDNA genome encoding a therapeutic or reporter transgene.

33. A pharmaceutical composition comprising the nucleic acid delivery vector according to any of claims 1 to 32 and a pharmaceutically acceptable carrier or diluent.

34. A pharmaceutical composition according to claim 33, wherein the composition is acceptable for therapeutic administration.

35. A method of producing a nucleic acid delivery vector, the method comprising: expressing capsid proteins or portions thereof from a non-enveloped mammalian virus in prokaryotic cells; combining the expressed capsid proteins or portions thereof with a nucleic acid comprising: a nucleic acid payload; and a DNA packaging sequence of the non-enveloped mammalian virus, wherein the capsid proteins or portions thereof self-assemble and encapsidate the nucleic acid payload forming the nucleic acid delivery vector.

36. The method of claim 35, wherein the capsid proteins or portions thereof and the nucleic acid are combined in a 6:1 mass ratio (w / w) of capsid protein or portion thereof to nucleic acid.

37. The method of claim 35 or 36, wherein the DNA packaging sequence comprises the nucleic acid as set forth in SEQ ID NO: 20, SEQ ID NO:21 or SEQ ID NO:22.

38. The method of any of claims 35 to 37, further comprising a step of purifying the nucleic acid delivery vector.

39. The method of any of claims 35 to 38, wherein the method further comprises a step of purifying the capsid proteins or portions thereof before the step of combining.

40. The method of claim 39, wherein the capsid proteins or portions thereof are N-terminally tagged and the purifying is done by affinity chromatography.

41. The method of claim 39, wherein the capsid proteins or portions thereof are N-terminally His-tagged and are purified by Ni column purification.

42. The method according to any of claims 35 to 41 , wherein the step of combining occurs in vitro.

43. The method according to any of claims 35 to 42, wherein the nucleic acid comprising the nucleic acid payload and the DNA packaging sequence of the non-enveloped mammalian virus are expressed in different prokaryotic cells than the capsid proteins.

44. The method according to claim 42, wherein the capsid proteins or portions thereof and the nucleic acid comprising the nucleic acid payload and the DNA packaging sequence of the nonenveloped mammalian virus are combined in 1 * assembly buffer solution comprising 50 mM Tris- HCI, 50 mM NaCI, 10 mM KCI and 5 mM MgCI2with a pH of 7.2.

45. The method according to any of claims 35 to 44, wherein the step of combining includes an overnight incubation.

46. The method according to claim 45, wherein the overnight incubation is at 4 degrees Celsius.

47. The method according to claim 35, wherein the step of combining occurs in prokaryotic cells and the nucleic acid payload is encapsidated by the capsid proteins or portions thereof in vivo in prokaryotic cells .

48. The method of any of claims 35 to 47, further comprising a step of purifying the nucleic acid delivery vector.

49. The method of any of claims 35 to 48, wherein the prokaryotic cells are E.coli cells.

50. The method of any of claims 35 to 49, wherein the capsid protein or portion thereof comprises the N-terminal 3rd portion of a TTV19 ORF1 capsid protein having a molecular weight of 25 kDa.

51. The method of any of claims 35 to 50, wherein the DNA packaging sequence is the intergenic region of a TTV19 anellovirus.

52. The method of any of claims 35 to 51 , wherein the DNA packaging sequence comprises the nucleic acid as set forth in SEQ ID NO: 20, SEQ ID NO:21 or SEQ ID NO:22.

53. A method of producing a nucleic acid delivery vector, the method comprising: co-transforming prokaryotic cells with a nucleic acid sequence encoding a capsid protein or portion thereof and a nucleic acid encoding a nucleic acid payload and a DNA packaging sequence; assembling nucleic acid delivery vectors encapsidating the nucleic acid payload in the prokaryotic cells; and optionally, purifying assembled nucleic acid delivery vectors encapsidating the nucleic acid payload.

54. A method of producing TTV19 nucleic acid delivery vectors that yields >1O10viral particles / mL at the harvest stage, comprising: expressing TTV-19 capsid proteins or portions thereof from a non-enveloped mammalian virus in prokaryotic cells; combining the expressed capsid proteins or portions thereof with a nucleic acid comprising: a nucleic acid payload; and a DNA packaging sequence of the TTV19 virus, wherein the TTV19 capsid proteins or portions thereof self-assemble and encapsidate the nucleic acid payload forming the nucleic acid delivery vector.

55. The method according to claim 54, wherein the step of combining occurs in vitro.

56. The method according to claim 55, wherein the method further comprises an optional step of purifying the TTV19 capsid proteins or portions thereof prior to combining them with the nucleic acid.

57. The method according to claim 54, wherein the step of combining occurs in vivo, wherein the nucleic acid is expressed in the same prokaryotic cells as the TTV-19 capsid proteins or portions thereof.

58. The method according to any of claims 54-57, wherein the method comprises a further step of purifying the nucleic acid delivery vector.

59. A nucleic acid delivery vector produced by the method of any of claims 35 to 58.

60. A cell line or helper system that supports the assembly and replication in vitro of a nucleic acid delivery vector according to any of claims 1 to 32 or a nucleic acid delivery vector produced by the method according to any of claims 35 to 46 and 48-52 or 54-56.

61. A scalable process to purify and isolate nucleic acid delivery vectors according to any of claims 1 to 32 or a nucleic acid delivery vector produced by the method according to any of claims 35 to 58 using rapid-sequential chromatography, wherein the isolated nucleic acid delivery vectors are high-titer and endotoxin free.

62. A use of the nucleic acid delivery vector according to any of claims 1 to 32, the pharmaceutical composition of claims 33 or 24, or a nucleic acid delivery vector produced by the method according to any of claims 35 to 58 for delivery of the nucleic acid payload to a cell.

63. A use of the nucleic acid delivery vector according to any of claims 1 to 32, the pharmaceutical composition of claim 33 or 34, or a nucleic acid delivery vector produced by the method according to any of claims 35 to 58 for gene therapy.

64. A use of the nucleic acid delivery vector according to any of claims 1 to 32, the pharmaceutical composition of claim 33 or 34, or a nucleic acid delivery vector produced by the method according to any of claims 35 to 58 for immune-stealth gene delivery to hematopoietic, hepatic, pulmonary, ocular or mucosal tissues.

65. A use of the nucleic acid delivery vector according to any of claims 1 to 32, the pharmaceutical composition of claim 33 or 34, or a nucleic acid delivery vector produced by themethod according to any of claims 35 to 58 for repeat-dose systemic or localized gene therapy without eliciting adaptive immune neutralization.

66. A kit for producing a vector, the kit comprising: a DNA vector plasmid comprising: a viral DNA binding sequence; and a nucleic acid payload, wherein the viral DNA binding sequence and the nucleic acid payload are flanked by an M13 f1 origin of replication START sequence and an M13 f1 origin of replication STOP sequence; a helper plasmid for producing and packaging single stranded DNA containing the nucleic acid payload, the helper plasmid comprising all the genes required for M13 replication, transcription and packaging; and a plasmid for expressing ORF1 capsid proteins or portions thereof in prokaryotic cells .

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