Production of Double-stranded DNA Ministrings

Abstract

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ministrings possess a heightened safety profile and also efficiently deliver DNA cargo to targeted cells. Conventional DNA vectors carry undesirable prokaryotic sequences, including antibiotic resistance genes, CpG motifs, and bacterial origins of replication, which may lead to the stimulation of host immunological responses. The bioavailability of conventional DNA vectors is also compromised due to their larger molecular size. Their circular nature may also impart chromosomal integration, leading to insertional mutagenesis.Bacterial sequences are excised from DNA minivectors, leaving only the gene of interest (GOI) and necessary eukaryotic expression elements. Our LCC DNA minivectors, or DNA ministrings, are devoid of immunogenic bacterial sequences; therefore improving their bioavailability and GOI expression. In the event of vector integration into the chromosome, the LCC DNA ministring will lethally disrupt the host chromosome, thereby removing the potentially dangerous mutant from the proliferating cell population. Consequently, DNA ministrings offer the benefits of 'minicircle' DNA while eliminating the potential for undesirable vector integration events. In comparison to conventional plasmids and their isogenic circular covalently closed (CCC) counterparts, DNA ministrings demonstrate superior bioavailability, transfection efficiency, and cytoplasmic kineticsthey thus require lower amounts of cationic surfactants for effective transfection of target cells.We have constructed a one-step inducible in vivo system for the production of DNA ministrings in Escherichia coli that is simple to use, rapid, and scalable.

Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells

“…Currently, the TelN- tos module has been successfully used to linearize engineered DNA vectors up to 110 kb and natural E. coli genome up to 4.64 Mb . Since linear artificial chromosomes with telomeres more closely represent the structure of natural mammalian chromosomes, the use of tos containing DNA vectors may provide an alternative choice for gene therapy and genomic studies. , It was shown that TelN-linearized DNA vectors can circumvent the unstable cloning of large or repetitive DNA sequences that are unclonable in traditional circular plasmids as well as provide an efficient in vivo or in vitro production of therapeutically useful miniDNA vectors without bacterial backbone . Additionally, adaptation of TelN- tos module into mammalian cells can facilitate the replication of DNA vectors in transfected cells, , suggesting their possible use in transfection studies for long-term transgene expression.…”

“…It was previously shown that linear miniDNA offers several advantages in gene delivery over conventional plasmids. Generally, miniDNA without bacterial backbone holds two major advantages: (i) smaller size that contributes to better bioavailability and more resistant to DNA shearing, leading to improved gene delivery and transgene expression; 60,61 and (ii) higher immuno-compatibility and less prone to transgene silencing in host cells as the immunogenic bacterial backbone is removed. 60,62 applied in the development of DNA vaccines for viral 63 or parasitic 64 infections, and immunotherapy against cancers 65 Due to the presence of the closed hairpin ends, TelNlinearized miniDNA offers the additional advantage of having a much lower frequency of natural and targeted genomic integration events than circular or opened-ended linear miniDNA in bacterial 50 and mammalian 66 cells.…”

Engineering DNA vaccines against infectious diseases

“…It has been reported that the FDA recommends integration studies, regardless of the delivery method, if there are greater than 10,000 copies of foreign DNA per microgram of host DNA [71]. Interestingly, minicircle DNA [51,53,54,72,73] and ministring DNA [74,75] have been reported to have less risk of insertional mutagenesis because the major bacterial DNA (i.e., the unmethylated CpG repeats functioning as PAMPs) is removed.…”

A Guide to Nucleic Acid Vaccines in the Prevention and Treatment of Infectious Diseases and Cancers: From Basic Principles to Current Applications

“…Such mutations can cause a gene to dysfunction or inactivate (i.e., a tumor suppressor gene) ( Würtele et al, 2003 ). It is reported that minicircle DNA and ministring DNA had less risk of insertional mutagenesis due to the removal of the major bacterial DNA (i.e., the unmethylated CpG repeats functioning as PAMPs), which suggested that such non-viral gene delivery vectors not only provided enhanced transgene expressions, but also were safer with less risk of insertional mutagenesis ( Chen et al, 2003 ; Kay et al, 2010 ; Sum et al, 2015 ; Colluru et al, 2016 ; Munye et al, 2016 ; Wong et al, 2016 ; Monjezi et al, 2017 ).…”