Useful background information

The pDG plasmids for the production of AAVs form a 2-plasmid transfection system. This means that in contrast to triple transfection with three different plasmids, only two components are needed: A plasmid with the gene of interest to be transferred, flanked by ITRs (vector plasmid) and the corresponding pDG plasmid containing all the information for the propagation and packaging of the gene of interest (the so-called helper & packaging plasmid). Additional infection with helper viruses – required in the original systems – is not necessary as the Adenovirus helper functions are included in the pDG plasmids.

Various so-called serotypes, created by modification of the binding motif on the AAV capsids, allow for targeted and efficient transfection of cells or tissues and additionally increase infection efficiency due to reduction of the potential for recognition by serum antibodies of the immunsystem and with this transgene expression in the target cell.

AAVs are currently gaining importance in gene therapy for the delivery of genetic material into the cell. AAV will cause no viral disease symptoms in the target organism, and only a mild immune response is to be expected. Integration into the host genome as achieved by transfection with lentiviral vectors (LV) is extremely rare.

Antibiotics are agents that kill or inhibit bacterial growth when added to the growth media. In order to selectively grow bacteria carrying a specific plasmid, genes are integrated on the plasmid that convey a resistance against a certain agent. As in this scenario, the plasmid provides a benefit, the resistance gene ensures that the plasmid persist within the bacterial population. However, the use of antibiotics and antibiotic resistance genes bears certain risks. Horizontal gene transfer may lead to broader distribution of the antibiotic resistance or, depending on the application, the resistance gene might also be transferred into the patient’s genome. Thus, alternative selection systems or strategies will become more important in near future. In its guidelines for medical gene transfer products, the European Medicines Agency (EMA; formerly European Agency for the Evaluation of Medicinal Products, EMEA) recommends abandoning the use of selection markers, such as resistance to antibiotics, in plasmid DNA products (CPMP/BWP/3088/99). In the interest of increased safety, national bodies (e.g. Paul-Ehrlich-Institut in Germany; AFSSAPS in France; and MCA in the United Kingdom) have adopted this recommendation. In reaction, PlasmidFactory has developed a method for the reliable large-scale production of Minicircle DNA. This already meet future’s regulations due to the lack of selection markers.

PlasmidFactory uses only specific E. coli strains, characterized and optimized for our DNA manufacturing process. The patented procedure for the processing of plasmid-containing cells is based on the alkaline lysis procedure of the bacterial biomass, resulting from fermentation. A well-known problem with the subsequent purification by means of ordinary ion exchange chromatography (plasmid kits) consists of contamination with bacterial chromosomal fragments (often called “genomic DNA” which is inappropriate since plasmids do also form a part of the genome), which bind specifically or unspecifically to the column matrix, just as plasmid DNA does. In the further course of the plasmid extraction procedure, this bacterial chromosomal DNA is often not separated from the plasmid. This will result in a high degree of contamination with bacterial chromosomal DNA, which may on the one hand significantly skew plasmid concentration measurements and on the other induce immune responses of the target expression system, and thus, jeopardize the success of your experiments. PlasmidFactory’s CCC grade DNA qualities are virtually free of bacterial chromosomal DNA (see Figure below). In our other qualities (Research grade), the proportion of these unwanted contaminations is significantly reduced compared to kit-based systems. In addition, quantitative analysis of the bacterial chromosomal DNA (qPCR) is available to customers.

The biosafety level (BSL) system is a categorization system that is associated with a set of biocontainment precautions used to isolate dangerous biological agents within enclosed laboratory facilities. These levels range from the lowest, BSL-1, to the highest, BSL-4. They ensure safety for researchers, the environment, and the public.

The capillary gel electrophoresis (CGE) is the only reliable tool to analyse different plasmid topologies. Our unique CGE technology allows determination and quantification of the proportion of the ccc form. CGE service can be ordered optionally for every plasmid production or independently for external DNA samples. It documents the degree of plasmid homogeneity – a parameter not only of practical interest for achieving optimum transfection results but also required from a pharmaceutical perspective. The main contaminant of kit-based plasmid DNA – bacterial chromosomal DNA – cannot be quantified by this method.

The CMV promoter, also known as the human CMV major immediate-early promoter, is derived from the human cytomegalovirus (CMV). It is one of the most widely used promoters in expression and drives constitutive gene expression in mammalian cells.

Cytosine-phosphate-guanosine nucleotides (CpG) in eukaryotic genomes are often methylated by DNA methyltransferases. In bacteria, this mechanism is used for delimitation of gene products. A negative consequence of such methylation can be a reduced expression rate of the affected gene. Avoiding such motifs in the vector system, e.g. by the use of minicircle DNA, is therefore advantageous.

PlasmidFactory has obtained an exclusive, worldwide license for the production, distribution and use of the helper & packaging plasmids of the pDG and pDP families from the German Cancer Research Centre (DKFZ).

The EF1α promoter, also known as the human EF1α promoter, is derived from the human elongation factor 1α gene. It serves as a constitutive promoter that drives high levels of gene expression in various cell types.

The light emission of fireflies occurs due to the action of the enzyme firefly luciferase on a molecule called luciferin. Firefly luciferase catalyzes the oxidation of luciferin, requiring oxygen and ATP. The enzyme has been used in biotechnology for reporter assays or imaging.

GFP is a protein that exhibits green fluorescence when exposed to light in the blue to ultraviolet range and was first isolated from Aequorea Victoria. In cell and molecular biology, the GFP gene is frequently used as a reporter of gene expression or transfection efficiency.

GMP refers to a set of guidelines and regulations that ensure the production, storage and distribution of drugs, biologics, and other pharmaceutical products meets highest quality standards. Compliance with GMP is essential for regulatory approvals by agencies like the EMA and FDA.

A gene of interest is a segment of DNA that encodes a specific product, such as a protein or RNA molecule.

HEK293 cells, also known as human embryonic kidney 293 cells, are an immortalized cell line derived from human embryonic kidney cells. These cells were isolated from a female fetus in the 1970s and have been widely used in research due to their reliable growth and transfection capabilities. HEK cells serve as hosts for gene expression experiments and protein production.

An IRES (Internal Ribosome Entry Site) is an RNA sequence element that allows for translation initiation in a cap-independent manner. Researchers use IRES elements as a powerful tool for co-expressing multiple genes from a single transcriptional unit in genetic vectors.

ITRs are symmetrical DNA sequences found at the ends of certain viral genomes. Each ITR presents the sequence of the other in a reverse complement manner. These sequences play a crucial role, e.g., in AAV production. ITRs serve as recognition sites for the AAV Rep proteins, enabling the formation of the viral replication complex. This complex initiates DNA replication and ensures the production of sufficient viral genomes for packaging. Moreover, ITRs act as packaging signals, guiding the encapsidation of the AAV genome into the viral capsid during production. Production and retainment of intact ITRs is vital for the successful assembly of functional AAV particles.

Lipopolysaccharides (LPS), often referred to as endotoxins, are produced by gram-negative bacteria such as E. coli and detected by the immune system of eukaryotic organisms. This can trigger an immune response and result in reduction of gene expression. In kit preparations, LPS content may vary very widely and have undesirable effects on the results of the experiment. During each plasmid production, PlasmidFactory measures the LPS content and can guarantee, upon request, a client specific content. Thus, the levels undermatch those of all other manufacturing processes.

Minicircles are Next Generation Gene Vectors consisting almost exclusively of the gene of interest (GOI) and a very short sequence which inevitably remains in the minicircle (SCAR). During Minicircle production intramolecular recombination in E. coli yields two circular DNA structures – the minicircle and a miniplasmid. In comparison to typical plasmid DNA with the general portion known as the bacterial backbone, comprising the genetic marker for selection (e.g. by antibiotics) and the so-called origin of replication (ori; for the replication of the plasmids in E. coli cells), minicircles are extremely small – down to half the size of the smallest plasmids. The dissemination of antibiotic resistance genes and the uncontrolled expression of backbone sequences may have a strongly negative influence on gene expression and could elicit immune response of the target cells. In addition, it was shown that unmethylated CpG motifs contribute to the silencing of episomal genes. In vector development, it is, therefore, an important objective to produce supercoiled (ccc) minicircle DNA without bacterial sequences. The used production method leading to a highly homogeneous minicircle product is proprietary to PlasmidFactory which produces both standard minicircle (In Stock products) as well as customized minicircle DNA.

The origin of replication (ORI), also known as the replication origin, is a specific sequence within a genome where DNA replication begins. It serves as the starting point for accurate duplication of genetic material during cell division. The origin of replication significantly influences the copy number of the plasmid in the bacterium.

Plasmids are small, typically ring-shaped, autonomously replicating, double-stranded DNA molecules found in bacteria and archaea. They exist as extrachromosomal genetic element. Nowadays, they play a crucial role as versatile gene vectors in molecular biology and biotechnology.

Plasmids can assume various conformations (see figure below): Intact plasmids are covalently closed circular (ccc-form) and negatively supercoiled. Single strand breaks due to enzymatic activity or mechanical stress lead to a relaxation of the DNA molecule. The resulting form is the so called open circular (oc) topology. Restriction endonucleases digest the DNA at certain positions resulting in the linear form. Moreover, mistakes in the cellular replication procedure and homologous replication lead to multimeric plasmid forms. These multimers are e. g. concatemers of two or more monomer forms, where also ccc, oc and linear forms can be observed. The only reliably active conformation is ccc. The different topologies can be visualized using agarose gel electrophoresis (AGE) with subsequent staining of the DNA bands using a fluorescent dye. However, the identification of the respective bands is difficult because the mobility of the different structures changes during the electrophoretical procedure. For most plasmids the ccc monomer is the fastest moving form. The oc monomer and ccc dimer very often appear as one band and cannot be distinguished by AGE. Here, the capillary gel electrophoresis (CGE) is the only reliable tool.

Retropackaging occurs during AAV production in HEK cells when sequences are inadvertently packed into an AAV particle that are not located between the ITRs, but outside such as bacterial backbone sequences. This is not intentional and bears certain risks, e.g. increased immunogenicity. The risk of retropackaging can be greatly reduced by using minicircles for the production of AAVs. These do not carry any backbone sequences but only the GOI and a short SCAR sequence.

S/MAR elements (scaffold/matrix attachment regions) are anchor sequences by means of which independently regulatable chromatin domains are linked to the protein skeleton of the nucleus. In addition to support of transcription and protection of (foreign) genetic information against inactivation, a special feature of these S/MAR elements has gained particular importance in the production of highly efficient plasmid DNA: Plasmids which comprise, in addition to the (e.g. therapeutically) effective gene, an S/MAR-element inserted at a defined location are replicated episomally and passed on as independent units to the daughter cells of the eukaryotic target cell. In other words, the plasmid is maintained as an episome for many generations, even in the absence of selection pressure, and will be stably expressed as an autonomous genetic unit without integrating. By now, the anchor sequence has been successfully reduced to a minimal motive of about 70 base pairs without significant reduction of the stable propagation to the daughter cells. Corresponding plasmids and minicircles with S/MAR elements for stable gene expression are known, e.g. as the pEPI or pEpito families, and marketed by PlasmidFactory on the basis of corresponding licensing agreements.

The sequence for chromatography, affinity and recombination is the sequence inevitably remains from the intramolecular recombination during minicircle production in E. coli and is not part of the GOI.

PlasmidFactory has developed a special production process for plasmid DNA (CCC grade classic) which includes the exclusive use of raw materials according to EMA/410/01 rev.3, 2011/C 73/01 (TSE/BSE certificate). Avoiding use of animal-based substances is of importance for clinical applications, since these substances may pose a safety risk. In addition, the use of enzymes is avoided completely during the manufacturing process by removing the RNA chromatographically. At PlasmidFactory, E. coli cultures to amplify plasmid DNA are always carried out in a fermenter and under controlled conditions, because it is not possible to guarantee the reproducibility of the produced biomass when shake flasks are used. The quality of the product is constantly monitored and documented.

A transgene is a segment of DNA containing a gene sequence that has been isolated from one organism and introduced into a different host organism. This non-native DNA segment may either retain the ability to produce RNA or protein in the transgenic organism or modify the normal function of the recipient organism’s genetic code.

The vector plasmid carries the transgene that is meant to be transferred to the target cell flanked by ITRs. It is one of the components needed to produce AAVs.

A PolyA stretch is a sequence consisting of consecutive adenins and can vary in length. In eucaryotic cells, mRNA molecules are polyadenylated post-transcriptionally to enhance mRNA stability and translation efficiency. Having the polyA tail encoded in the plasmid, facilitates template production and gene expression in the eukaryotic host cell.

β-Galactosidase catalyzes the hydrolysis of glycosidic bonds in β-Galactopyranosides. In microbiology and molecular biology, the lacZ gene from E. coli (encoding β-Galactosidase) serves as a reporter gene as the hydrolysis of X-Gal (5-Bromo-4-chloro-3-indoxyl-β-D-galactopyranoside) produces a blue, water-insoluble indigo dye. Like this, the temporal expression of genes can be analyzed.