production-of-double-stranded-dna-ministrings

Production of Double-stranded DNA Ministrings

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ...

University of Waterloo

Different kinds of mutations, mostly point mutations, in the coagulation factor IX (FIX) gene F9 result in a recessive X-linked bleeding disorder known as haemophilia B. In this study, molecular analysis of 76 unrelated Iranian haemophilia B patients was performed by PCR, single strand conformational polymorphism (SSCP) on important functional regions of the F9 gene followed by sequencing on samples with different migration pattern. Using this approach we found mutation in 52 out of 76 patients. Our data showed that the pathologic mechanisms are heterogeneous as recorded for patients in haemophilia B mutation database and seven of the mutations are previously undescribed.

Identification of factor IX mutations in Iranian haemophilia B patients by SSCP and sequencing.

The P1 plasmid partition system depends on ParA-ParB proteins acting on centromere-like parS sites for a faithful plasmid segregation during the Escherichia coli cell cycle. In vivo we placed parS into host E. coli chromosome and on a Sop(+) F plasmid and found that the stability of a P1 plasmid deleted for parA-parB could be partially restored when parB was expressed in trans. In vitro, parS, conjugated to magnetic beads could capture free parS DNA fragment in presence of ParB. In vitro, ParA stimulated ParB-mediated association of intermolecular parS sites in an ATP-dependent manner. However, in the presence of ADP, ParA reduced ParB-mediated pairing to levels below that seen by ParB alone. ParB of P1 pairs the parS sites of plasmids in vivo and fragments in vitro. Our findings support a model whereby ParB complexes P1 plasmids, ParA-ATP stimulates this interaction and ParA-ADP inhibits ParB pairing activity in a parS-independent manner.

ParAB-mediated intermolecular association of plasmid P1 parS sites.

While safer than their viral counterparts, conventional non-viral gene delivery DNA vectors offer a limited safety profile. They often result in the delivery of unwanted prokaryotic sequences, antibiotic resistance genes, and the bacterial origins of replication to the target, which may lead to the stimulation of unwanted immunological responses due to their chimeric DNA composition. Such vectors may also impart the potential for chromosomal integration, thus potentiating oncogenesis. We sought to engineer an in vivo system for the quick and simple production of safer DNA vector alternatives that were devoid of non-transgene bacterial sequences and would lethally disrupt the host chromosome in the event of an unwanted vector integration event.

Construction and characterization of an in-vivo linear covalently closed DNA vector production system.

Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed.

Bacteriophage recombination systems and biotechnical applications.

While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage λ pL promoter and regulated by the thermolabile λ CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called "Super Sequences" that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system, achieving an overall LCC DNA minivector production efficiency of ∼ 90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics.

Optimization of a one-step heat-inducible in vivo mini DNA vector production system.

Conventional plasmid DNA vectors play a significant role in gene therapy, but they also have considerable limitations: they can elicit adverse immune responses because of bacterial sequences they contain for maintenance and amplification in prokaryotes, their bioavailability is compromised because of their large molecular size, and they may be genotoxic. We constructed an in vivo platform to produce ministring DNA-mini linear covalently closed DNA vectors-that are devoid of unwanted bacterial sequences and encode only the gene(s) of interest and necessary eukaryotic expression elements. Transfection of rapidly and slowly dividing human cells with ministring DNA coding for enhanced green fluorescent protein resulted in significantly improved transfection, bioavailability, and cytoplasmic kinetics compared with parental plasmid precursors and isogenic circular covalently closed DNA counterparts. Ministring DNA that integrated into the genome of human cells caused chromosomal disruption and apoptotic death of possibly oncogenic vector integrants; thus, they may be safer than plasmid and circular DNA vectors.

DNA ministrings: highly safe and effective gene delivery vectors.

Glaucoma is a neurodegenerative eye disease that causes permanent blindness at the progressive stage and the number of people affected worldwide is expected to reach over 79 million by 2020. Currently, glaucoma management relies on pharmacological and invasive surgical treatments mainly by reducing the intraocular pressure (IOP), which is the most important risk factor for the progression of the visual field loss. Recent research suggests that neuroprotective or neuroregenerative approaches are necessary to prevent retinal ganglion cells (RGCs) loss and visual impairment over time. Neuroprotection is a new therapeutic strategy that attempts to keep RGCs alive and functional. New gene and cell therapeutics encoding neurotrophic factors (NTFs) are emerging for both neuroprotection and regenerative treatments for retinal diseases. This article briefly reviews the role of NTFs in glaucoma and the potential delivery systems. For further resources related to this article, please visit the WIREs website.

Neuroprotective therapies in glaucoma: I. Neurotrophic factor delivery.

Neurotrophic factor genome engineering could have many potential applications not only in the deeper understanding of neurodegenerative disorders but also in improved therapeutics. The fields of nanomedicine, regenerative medicine, and gene/cell-based therapy have been revolutionized by the development of safer and efficient non-viral technologies for gene delivery and genome editing with modern techniques for insertion of the neurotrophic factors into clinically relevant cells for a more sustained pharmaceutical effect. It has been suggested that the long-term expression of neurotrophic factors is the ultimate approach to prevent and/or treat neurodegenerative disorders such as glaucoma in patients who do not respond to available treatments or are at the progressive stage of the disease. Recent preclinical research suggests that novel neuroprotective gene and cell therapeutics could be promising approaches for both non-invasive neuroprotection and regenerative functions in the eye. Several progenitor and retinal cell types have been investigated as potential candidates for glaucoma neurotrophin therapy either as targets for gene therapy, options for cell replacement therapy, or as vehicles for gene delivery. Therefore, in parallel with deeper understanding of the specific protective effects of different neurotrophic factors and the potential therapeutic cell candidates for glaucoma neuroprotection, the development of non-invasive and highly specific gene delivery methods with safe and effective technologies to modify cell candidates for life-long neuroprotection in the eye is essential before investing in this field.

Neuroprotective therapies in glaucoma: II. Genetic nanotechnology tools.

In combination with novel linear covalently closed (LCC) DNA minivectors, referred to as DNA ministrings, a gemini surfactant-based synthetic vector for gene delivery has been shown to exhibit enhanced delivery and bioavailability while offering a heightened safety profile. Due to topological differences from conventional circular covalently closed (CCC) plasmid DNA vectors, the linear topology of LCC DNA ministrings may present differences with regards to DNA interaction and the physicochemical properties influencing DNA-surfactant interactions in the formulation of lipoplexed particles. In this study, N,N-bis(dimethylhexadecyl)-α,ω-propanediammonium(16-3-16)gemini-based synthetic vectors, incorporating either CCC plasmid or LCC DNA ministrings, were characterized and compared with respect to particle size, zeta potential, DNA encapsulation, DNase sensitivity, and in vitro transgene delivery efficacy. Through comparative analysis, differences between CCC plasmid DNA and LCC DNA ministrings led to variations in the physical properties of the resulting lipoplexes after complexation with 16-3-16 gemini surfactants. Despite the size disparities between the plasmid DNA vectors (CCC) and DNA ministrings (LCC), differences in DNA topology resulted in the generation of lipoplexes of comparable particle sizes. The capacity for ministring (LCC) derived lipoplexes to undergo complete counterion release during lipoplex formation contributed to improved DNA encapsulation, protection from DNase degradation, and in vitro transgene delivery.

Physical Characterization of Gemini Surfactant-Based Synthetic Vectors for the Delivery of Linear Covalently Closed (LCC) DNA Ministrings.

Gene therapy is becoming an influential part of the rapidly increasing armamentarium of biopharmaceuticals for improving health and combating diseases. Currently, three gene therapy treatments are approved by regulatory agencies. While these treatments utilize viral vectors, non-viral alternative technologies are also being developed to improve the safety profile and manufacturability of gene carrier formulations. We present an overview of gene-based therapies focusing on non-viral gene delivery systems and the genetic therapeutic tools that will further revolutionize medical treatment with primary focus on the range and development of non-invasive delivery systems for dermal, transdermal, ocular and pulmonary administrations and perspectives on other administration methods such as intranasal, oral, buccal, vaginal, rectal and otic delivery.

Nafiseh Nafissi

School of Pharmacy

University of Waterloo

Production of Double-stranded DNA Ministrings