Non Viral Transfection Of Mammalian Cells Biology Essay

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As 'process is product' is a key factor in the biopharmaceutical industry, the need of better techniques for DNA transfer is essential for the large-scale recombinant protein production in mammalian cells. Transient transfection in mammalian cells such as human embryonic kidney 293 or Chinese hamster ovary cells has been reported for the production of milligram to gram quantities of recombinant protein. Calcium phosphate precipitation (CaPi) and polyethyleimine (PEI) are the two most extensively used non-viral gene delivery systems. The highest transient yields of 80-100mg/L have been obtained by transfecting CHO cells with PEI of 25 kDa. Non-viral delivery systems have made significant progress in developing better expression vectors, choice of cell lines , culture medium, transfection parameters and process scale-up.

Introduction

Stable gene expression technologies have been widely employed for the production of recombinant proteins from the mammalian cells. Owing to the drawbacks of these techniques (investment in time, resources) and increasing demand of the recombinant proteins in the market, it has become evident to develop a technique that is faster, cheaper and more reliable. With the advent of the transient systems in 1990s, many works have been done for the production of milligram to gram quantities of protein. Furthermore, the system is developed for the evaluation of different proteins or variants among the same protein {Cho et al., 2003}.

The main features of the transient expression systems are: simple in construction of expression vectors, time-efficient: generate products in days, genetically stable and consistent, multiple processing, wide applicability among various host cells {Wurm et al., 1999}. As 'process is product' is a key factor in the biopharmaceutical industry, the need of better techniques for DNA transfer is essential for the large-scale recombinant protein production in mammalian cells. Many engineered viral vectors such as adenoviruses, alphaviruses, baculoviruses, lentiviruses etc. are used for developing large amounts of recombinant proteins but they conjoin drawbacks such as restricted tropism, low transfection capacity and high cost of production {Pham et al., 2006}.

Non-viral delivery systems have been developed such as calcium phosphate, electroporation, lipid reagents and reported higher transfection capability with adherent as well as suspension adapted mammalian cells{Wurm,1999}. Different cell lines used for non-viral mediated transfection are COS (African green monkey kidney) {Blasley et al., 1996}, HEK-293(Human embryonic kidney) {Jordan et al., 1998}, CHO (Chinese Hamster Ovary) {Preuss, 2000}, BHK (Baby hamster kidney) {Wurm, 1999}. Non-viral delivery systems has made significant progress in developing better expression vectors, choice of cell lines , culture medium, transfection parameters and process scale-up{Pham,2006}.

Non-viral Gene Delivery/Transfection

The key factors of an ideal gene delivery system are: protect DNA from nucleases, translocate DNA into the nucleus of the target cell and have no adverse effect {Gao et al., 2007}. Viral delivery systems that have been developed for transfer of naked DNA into the mammalian cells are not very effective as compared to non-viral systems as former generally translocate plasmid DNA into cytoplasm rather than nucleus and thus proved to be less effective or very little gene expression detected {lungwitz et al., 2005}. Therefore vehicles with nuclear translocation (coupled with DNA and promote entry into the cell) potential must be employed for large-scale production of the recombinant proteins.

There are two types of non-viral gene delivery systems carrier-free gene delivery (physical: needle injection, gene gun, ultrasound, electroporation) and synthetic vector based gene delivery (chemical: Synthetic polymers, lipids and proteins) {Gao et al., 2007}. Cationic polymers, cationic lipids, nuclear proteins (histones) have shown to be effective vehicles {Colosimo et al., 2000} {Haberland et al., 2005} but they are highly expensive for a large-scale production. Zauner et al. has compared transfection capacity of two liposomal complexes known as Lipofectamine against poly (l-lysine) (PLL) and found polycations have greater transfection capability {Godbey et al., 2001}.

Electroporation of plasmid DNA is also practised but this system requires development of more precision as it causes cell death by electrical discharge {Pham et al., 2006}.Calcium phosphate DNA co-precipitation and polyethyleneimine (PEI) DNA complexes are the most efficient vehicle systems because they are cost-effective and could be used for large-scale production of both adapted and suspension cell lines , also they transfer DNA by forming complexes which are taken up by the cells through endocytosis {Wurm et al.,1999}.

Table 1: Gene Delivery barriers and multicomponent design of Non-viral vectors {Khanna et al., 2007}

D:\rnai-tbl3.jpg

Calcium-phosphate ( CaPi) Precipitation

Graham and Van der Eb developed calcium-phosphate DNA co-precipitation method in 1973 and since then it has been the most efficient transfection system in mammalian cells {Pham et al., 2006}. Precipitation of calcium-phosphate is formed by nucleation or particle growth and depends on the saturation, at low supersaturation the particle growth exaggerates but due to undersaturation the particle redissolves {Jordan et al., 2004}. Thus calcium phosphate method is dexterous and dynamic in nature, causes the change in characteristics of the particle by the continuous action of DNA on the precipitate {Coonrod et al., 1997}.

CaPi transfection generally involves a two-step protocol:

Adding DNA to calcium chloride solution

Furthermore, mixing it with phosphate to form calcium phosphate DNA precipitates

Adding complexes to the culture medium {Graham et al., 1973}.

The CaPi DNA complexes enter the target cells by the process of phagocytosis and then high calcium concentration causes the rupture of the vesicles. It has been found that plasmid DNA translocate into the nucleus within 1h of transfection {Coonrod et al., 1997}. Also work has been done to show transfer of 1000s of plasmid DNA into individual cell using calcium phosphate transfection. The concentration of the calcium phosphate is the main factor that effects its precipitate formation but other parameters such as temperature, pH and DNA concentration are also important {Jordan et al., 1996}. The table below describes the critical parameters that affect CaPi precipitate formation.

Table 2: Critical factors effecting calcium phosphate precipitate {Jordan et al., 2004}.

Polyethylenimine (PEI)

In contrast of other non-viral vectors, polymers have been widely used for the transfection of mammalian cells due to their characteristics of easy preparation, purification and chemical stability {lungwitz et al., 2005}. PEI is highly polycationic synthetic polymer that can be derived by condensation of 2-ethyl-2-oxazoline monomers or azridine and is available in linear or branched structures respectively. PEI is extensively used for its characteristics of low cost, potential for operation at scale, minimal cytotoxity and efficient transgene expression.

The branched PEI can be used for plasmid DNA delivery as well as for RNA or oligonucleotides and intact ribozymes {Aigner et al., 2002}. The efficiency of bPEI- derived vectors depends upon their characteristics of molecular weight, degree of branching, the cationic charge density, buffer capacity, polyplex capacity of DNA binding, content, size and absence or presence serum while transfection {lungwitz et al., 2005}.

The bPEI with higher molecular weight shows higher transfection capability along with stable complex formation as compared to the bPEI with lower molecular weight {Godbey et al., 1999}. With the increase in N/P ratios increases the net positive charges on the complexes thus help in cell interactions and nuclear uptake {Oh et al., 2002}.

Fig: 1 Synthesis of branched polyethylenimine by condensation of azridine in aqueous solution {Harpe et al., 2000}.

Linear PEI (lPEI) distinguishes itself from the branched PEI from the nuclear uptake transfection efficiency and toxicity. It is synthesized by polymerization of either unsubstituted or two substituted 2 oxazolines with acid or base hydrolysis of N-substituted polymer (Fig2) {Brissault et al., 2003}. It has been observed with increasing quantities of amino groups in the copolymer also effects the buffer capacity and transfection efficiency of the lPEI complexes, also large particles of the lPEI shows higher interaction with cell surfaces {lungwitz et al., 2005}. In comparison with bPEI, lPEI showed improved cell viability, promote nuclear localization and increased transfection efficiency {Zou et al., 2000}.

Fig2: Synthesis of linear polyethylenimine {Brissault et al., 2003}.

The intracellular trafficking pathways have revealed the understanding of the PEI- mediated gene transfer. Plasmid DNA packed with PEI is transferred to the cell by receptor-mediated endocytosis and thus can be used for labelling polyplexes with targeting moieties (Fig3). Cationic polyplexes interact with the negatively charged membrane glycoproteins, proteoglycans, and sulphated proteoglycans and start cell internalization via adsorptive or fluid-phase endocytosis {lungwitz et al., 2005}. The reactivity between cell and polyplex could be enhanced by increasing the net positive charge on the surface of the polyplex (change in concentration or incubation time) {Bieber et al., 2002}. The transfection of cell by PEI-DNA polyplexes starts within 3h after their incubation to the medium {Kristensen et al., 2001}.

The PEI-DNA complexes succeed through the endolysomal pathway and escapes efficiently endosomes/lysosomes by the 'Proton Sponge' effect by which protonable amino nitrogen present at every third atom of PEI buffer the endosomal environment and hence arrest acidification and fusion with the lysosomes and eventually swell the vesicles to release plasmid DNA into the cytoplasm {Akinc et al., 2005}. PEI-DNA complexes have shown high gene expression resulted in understanding of the fact that it generally transfers plasmid DNA from cytoplasm to the nucleus {Pollard et al., 1998}. The rate of PEI-mediated transfection and transgene expression depends upon the cell-type and cell culture medium {Kristensen et al., 2001}.

Experiments have been performed to understand the mechanism of protection of DNA by PEI and it has been stated that it protects DNA from degradation by DNase1 and DNase 2, protecting the DNA from degradation would lower gene expression level so protecting it while delivery is an important parameter {Godbey et al., 2001}.

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Fig3: Schematic representation of the gene transfer using ligand-decorated non-viral vectors {Kircheis et al., 2001}.

Transient (Non-Viral) Transfection of Mammalian Cells

Transient gene expression is a rapid method of direct conversion of recombinant DNA into recombinant protein. It has the following advantages over stable gene expression:

Expression over few days/weeks

Transfect in many copies of DNA

Transcription without integration into host genome

No subculturing

No selection for maintenance of expression vector

Quickly generates protein for protein assays, characterization etc.

Rapid for milligram to gram amounts

Cost effective

For large scale transient production of recombinant protein in mammalian cells, the key factors that should be taken into account are:

The cell line

The expression vector

The plasmid DNA quality

The transfection vehicle

The culture medium {Durocher et al., 2002}.

Large scale recombinant protein production depends upon the transfection capacity and for the development of such system requires the plasmid DNA expressing reporter proteins like secreted alkaline phosphatase (SEAP) and green fluorescent protein (GFP){Pham et al., 2006}.

Mammalian cell lines

For the efficient yield of recombinant protein the cell line should be easily transfected. Many different cell lines have been used in past such as COS and CV1 derived from African green monkey for the small scale transient production. The main drawback with these cells is that they show less productivity in contrast with other cell lines. Nowadays, the cell lines that are industrially accepted for the production of recombinant protein are Chinese hamster ovary (CHO), mouse myeloma, baby hamster kidney and human embryonic kidney 293 (HEK293).

Chinese Hamster Ovary (CHO)

CHO cell line is widely used for the production of recombinant proteins using stable gene expression but less work has been done with transient transfection. Initial work with these cell lines using CaPi-mediated transient transfection has not shown satisfying results as but later work with PEI- mediated transient transfection has given improved productivity of upto 10mg/L for antibodies {Wurm et al.,1999}. CaPi requires serum and osmotic shock for the production of high levels of protein but show low level of gene delivery with suspension-adapted CHO cell lines {}.

The highest transient yields of 80-100mg/L have been reported by transfecting CHO cells with linear PEI (25 kDa) {Wulhfard et al., 2008, 2010} {Ye et al., 2009}. In 2004 Derouazi et al. has worked on the serum free suspension adapted CHO cells and scaled up the process in 20L stirred bioreactor for the production of recombinant protein using PEI-mediated gene delivery (Fig.4) {Derouazi et al., 2004}.Recently Rajendra et al. has reported volumetric productivity of recombinant antibody of upto 300mg/L by optimizing the transient transfection parameters in suspension-adapted CHO cells {Rajendra et al., 2011}.

Fig4: Transfection of CHO cells in 3L and 20L bioreactor transfected at a DNA: PEI ratio of 1:2 {Derouazi et al., 2004}.

Human Embryonic Kidney 293

Human embryonic kidney 293 cell line is derived from transformed HEK cells with DNA of adenovirus 5 {Graham et al., 1977}. These cells have been widely used for the production of recombinant protein using transient gene expression as they are highly susceptible to most gene delivery systems and can grow in serum free suspension adapted medium. There are two different types of HEK 293 cells namely 293E (express EBNA1) and 293T (express SV40 large T-Ag) but both support episomal replication. The SV40 ori Tag system in 293 T cells does not promote high levels of transient production in these cell lines because of simultaneous occurrence of two process of gene replication and expression {Pham et al., 2006}. In contrast 293E cell lines are widely developed for large scale transient production using EBV oriP plasmids {Pham et al., 2003, 2005}. HKB11 is a new hybrid cell line developed by fusing 293 with Burkitt's lymphoma cell line and reported 20 fold high transient gene expression using oriP/EBNA1 based plasmid {Cho et al., 2003}.

CaPi and PEI are extensively used gene delivery systems in HEK-293 cell line and former has been utilized for scaled up to 100L in bioreactors {Baldi et al., 2005}. Durocher et al. used model proteins such as SEAP and GFP to design expression vector and monitor transfection efficiency in 293E cell using 25kDa PEI (Fig5) {Durocher et al., 2002}.

Fig 5: Transfection of different HEK293 cell lines with DNA: PEI ratio at 1:2 for SEAP expression and transfection efficiency {Durocher, 2002}.

Expression Vectors

They play a significant role for retrieving high yields of r-proteins using transient gene expression. CMV promoter is the most exploited among the mammalian cell lines, Xia et al. have compared different human, rat and mouse CMV promoters and reported highest expression with the hCMV in 293E and CHO cells {Xia et al., 2006}. Other promoter expressing elongation factor 1α (EF1 α) is also used in the CHO and HEK293 cell lines and has been reported more active than mCMV/EF1 promoter in Cho cells {Derouazi et al., 2004}. A hybrid metallothionein (MT) promoter has been shown to be more active than intron-less promoters (hCMV and SV40) in CHO cells {Pham et al., 2006}. EBV based vectors show improvement in large scale TGE by binding EBNA1 to the region of oriP, also Cachianes et al. reported 10 fold higher expression in 293 cells due to presence of oriP {Cachianes et al., 1993}.

For the production of heterodimeric recombinant proteins such as antibodies it is necessary to cotransfect two vectors (coding separate polypeptides) to obtain complete antibody molecule, thus Baldi et al. performed experiments using different ratios of two vectors pEAK-LH39 and PEAK-LH41 in suspension HEK 293 cells with the Ca-Pi transfection method ( Fig 6) {Baldi et al., 2005}.

Fig 6: Cotransfection of HEK293 cells with heavy(H) and light(L) chain coding DNA vectors using CaPi method, the highest antibody expression was observed with equal H/L ratio {Baldi et al., 2005}.

Plasmid DNA Amount

As reported a 45 L scale transfection generally requires 45 or 56 mg of plasmid DNA for CaPi or PEI methods, thus plasmid DNA could be generated by using 2-4 L of E.coli culture medium {Pham et al., 2006}. Extraction of plasmid DNA could be done by alkaline lysis protocol postulated by Birnboim and Dolly {Birboin et al., 1979}. Purification of supercoiled DNA is generally done by size exclusion and ion exchange chromatography.

Fig 7: A. Change in transfection efficiency C. Antibody expression level with varying amount of DNA as PEI was kept constant in CHO cells {Rajendra et al., 2011}.

Transfection (Non-viral) Vehicles

CaPi

Calcium phosphate precipitation is dynamic and reversible in nature and thus depends upon several critical parameters such as cell density, concentration of DNA, calcium phosphate, pH of the phosphate solution and time of medium dilution { Jordan et al., 2004}. The main drawback with CaPi-mediated transfection is its dependence on serum and requirement of media replacement before transfection; due to these factors it is difficult to transfect suspension adapted CHO cells {Derouazi et al., 2004}.

Another approach similar to CaPi precipitation has been developed called 'Calfection' but it still requires serum for the efficient production of r-proteins. Jordan et al. showed the effect of phosphate concentration in the precipitation mixture and reported that higher concentration of phosphate has a negative effect on transfection efficiency (Fig 8) {Jordan et al., 2004}.

Fig8: Effect of phosphate concentration on turbidity, DNA binding and recombinant protein expression level for adherent CHO and adherent HEK cells {Jordan et al., 2004}.

PEI

PEI-DNA polyplexes are the most efficient non-viral gene delivery system widely used among various mammalian cell lines for the production of high level of protein expression. It performs scaleable transfection process and thus provides recombinant protein yields in equivalence to CaPi (Table-3) {Baldi et al., 2005}.

Table-3: Comparison between PEI and CaPi-mediated transfection under serum-free conditions for the production of recombinant protein {Baldi et al., 2005}.

HEK293 cells transfected with PEI-mediated transient system showed that 25kDa branched PEI is more effective than rest of the molecular weights of PEI, also 25L PEI is more effective than 25B PEI for the transfection of suspension growing 293E cells {Durocher, 2002}. The highest yields have been reported by transfection of HEK-293 cells with 25 kDa linear PEI at a cell density of 20 x 106 cell/ml by subsequently addition of PEI and DNA to the medium containing valproic acid (VPA) {Backliwal et al., 2008c}.

Carpentier et al. has demonstrated the effect of amount of polyplex on the viable cell count, mRNA level and protein production in HEK293-EBNA1 cells transfected with 100%, 150% and 200% polyplex concentration and found that mRNA level was increased by using standard polyplex amounts (Fig9) {Carpentier et al., 2007}.

Fig 9: Effect of polyplex amount on viable cell count, mRNA level and protein production in HEK293-EBNA1 cells {Carpentier et al., 2007}.

Culture medium

Serum-free media is highly appreciated in the biopharmaceutical industry because it eradicates the potential viruses and thus makes the downstream processing of recombinant protein easy. But serum-free media has shown low transfection efficiency as compared to serum- containing media {Geisse et al., 2005}.

Negatively charged polyplexes interact with proteoglycans of membrane, as heparin present in serum free media causes the neutralization of these polyplexes and result in low transfection efficiency {Mislick et al., 1996}.

Durocher et al. demonstrated the effect of serum on transfection efficiency(GFP) and recombinant protein expression level(SEAP), transfection of suspension growing human2293- EBNA1 cells was done by both linear and branched PEI {Durocher et al., 2002}. The cells were added to the transfection mixture containing 1% serum and showed a 4-5 fold increase in protein expression level as compared to when added to the serum-free media; also with increase in concentration of serum from 1% to 5% it further showed improvement in transfection efficiency and protein production { Durocher et al., 2002}. In contrast when cells where added to the serum free medium they showed 50% less productivity as compared to the serum containing media.

Fig10: Effect of Serum on transgene expression {Durocher et al., 2002}.

Conclusion

Transient gene expression is a rapid method of direct conversion of recombinant DNA into recombinant protein. Large scale recombinant protein production depends upon the transfection capacity and for the development of such system requires the plasmid DNA expressing reporter proteins like secreted alkaline phosphatase (SEAP) and green fluorescent protein (GFP){Pham et al.,2006}.

Non-viral mediated transfection has resulted in production of milligrams to gram quantities of recombinant proteins. Calcium phosphate precipitation (CaPi) and polyethyleimine (PEI) are the most extensively used non-viral gene delivery systems in mammalian cells. The CaPi DNA complexes enter the target cells by the process of phagocytosis and then high calcium concentration causes the rupture of the vesicles whereas PEI-DNA complexes succeed through the endolysomal pathway and escapes efficiently endosomes/lysosomes by the 'Proton Sponge' effect.

CaPi and PEI are extensively used gene delivery systems in HEK-293 cell line and former has been utilized for scaled up to 100L in bioreactors {Baldi et al., 2005}. Critical factors of transfection vehicles such as pH, phosphate concentration, DNA concentration, also affect the overall recombinant protein productivity. Both CaPi and PEI perform scaleable transfection process and thus provide equal quantities of recombinant protein {Baldi et al., 2005}.Effect of serum is also significant in large scale transient production of recombinant protein and directly affects the transfection efficiency.

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