Vectors for Non-viral Gene Delivery - Clinical and Biomedical Applications

Review Article

Austin Therapeutics. 2015;2(1): 1014.

Vectors for Non-viral Gene Delivery - Clinical and Biomedical Applications

Venkata Kashyap Yellepeddi*

Department of Pharmaceutical Sciences, Roseman University of Health Sciences, USA

*Corresponding author: Venkata Kashyap Yellepeddi, Department of Pharmaceutical Sciences, Roseman University of Health Sciences, 10920 S River Front Parkway, Utah-84095, South Jordan, USA

Received: September 17, 2014; Accepted: February 27, 2015; Published: Accepted: February 27, 2015

Abstract

Use of viral vectors for effective delivery of therapeutic genes is frequently rebuffed by the body’s adaptive immune response against viral delivery vectors. Nonviral delivery system have been used to circumvent this problem but they are encountered with problems like transient expression and inflammatory responses induced by the reaction of the innate immune system reacting against bacterial DNA. However, within past decade the nonviral gene delivery has come to age and many efforts have been made to recognize the barriers for non-viral DNA delivery as potential allies in the development of novel therapeutics. This review summarizes important aspects of nonviral gene delivery such as barriers for gene therapy, various nonviral gene delivery vectors. The controlled release of pDNA using various stimuli responsive carriers have been discussed which shows a great potential to overcome barriers for gene delivery. Various clinical trials and patents involving nonviral vectors in gene therapy were briefly mentioned as the numbers show, there is an enormous growth of interest for nonviral gene delivery vectors. Novel strategies in nonviral gene delivery such as computer assisted intracellular kinetic models with kinetic parameters, fusogenic liposomes, conjugation of nuclear localization signals, were discussed which provide an impetus for researchers to achieve goal of preparing better nonviral transfecting agents with improved efficacy, enhanced stability and minimal toxicity.

Keywords: Gene therapy; Cationic liposomes; Transfection; Polyplexes; DNA condensation; Endosomal release

Introduction

In recent years gene therapy has become one of the highly publicized areas of biomedical, pharmaceutical and biotechnological research. In Gene therapy the normal gene or genetic material is used to replace the defective gene that is responsible for the disease. Scientific breakthroughs in the field of genomics and molecular biology have revealed that almost all diseases have a genetic component. Gene therapy has been considered not only one of the most potential approach for treatment of genetic disorders but also an alternative approach to deliver proteins, as currently they are manufactured by incorporating genes in microorganisms which are cultured in the laboratory that produce required proteins which were coded by the incorporated genes. Gene therapy has been one of the most enigmatic areas of research which shows a lot of promise for treating wide spectrum of diseases but at the same time has been suffered by setbacks and has become challenge to scientific community [1].

Gene therapy is considered a unique approach in therapeutics as it can be adapted towards treatment of both inherited and acquired diseases. Gene delivery involves encapsulation of a gene of interest, which is ideally intended for delivery into the target cells. After a series of events taking place such as uptake by endocytosis, release of DNA into the cells, transcription followed by translation the protein of interest is produced. In order to achieve successful gene delivery, significant barriers must be overcome at each step of the above mentioned events to optimize gene activity while minimizing the potential inhibitory inflammatory responses.

Since the initiation of first gene therapy clinical trial fifteen years back, there were several ups and downs which staggered the field of human gene therapy. The gene therapy roller coaster started in 1990 with the treatment of the first patient in a gene therapy clinical trial for Severe Combined Immuno Deficiency (SCID) using a retroviral vector ex vivo to introduce the deficient adenosine deaminase gene in autologous T-cells [2]. This gave impetus for scientific community and nearly up to a decade the field of gene therapy blossomed with the validation of nonviral gene medicines and genetic vaccines in a plethora of animal models using plasmid- based systems. It was during that period the first nonviral gene therapy clinical trial by intratumoral injection of a cationic lipid-formulated plasmid to cancer patients [3], and the first plasmid bases vaccine clinical trial for diseases like HIV, malaria, and flu were initiated, more the a hundred gene therapy biotech companies were created worldwide [4- 6]. Then a series of setbacks happened with first being happened in 1999 with the death of a patient following multiple organ failure in response to the intra hepatic infusion of an adenoviral vector. The second setback occurred in 2003 when two infants with X-linked severe combined immunodeficiency successfully treated with an ex vivo retroviral vector developed a T-cell leukemia like syndrome as a result of insertional mutagenesis [7]. In the early 2004 fortunately another clinical successes has triggered the interest of the public and stimulated the impetus and creativity of the scientific and medical community which was the world’s first human gene therapy product, Gendicine®, an adenoviral vector expressing the tumor- suppressor gene p53, obtained market approval by the Chinese FDA for the treatment of head and neck squamous cell carcinoma. The large part to the setbacks was contributed by the viral vectors thus improving the safety of viral vectors has been emphasized a lot. Although viruses have been modifies to produce viral vectors that would not cause disease but would still be able to efficiently transducer the host cells, several significant issues remain unanswered related to their safety and manufacturing. Even though the number of clinical trials using nonviral gene products is small (~20%) compared to viral vectors the number of publications on nonviral gene delivery has increased exponentially over the past few years and many new delivery systems are being investigated in pre-clinical research development [8]. Viral vectors offer unique advantages as specificity and efficiency of transfection when compared with non-viral vectors. However, viral vectors suffer from disadvantages such as over expression of genes, pathogenicity and immunogenicity [9,10].

Gene delivery strategies

The challenge of specific and efficient delivery of genetic material into the diseased sites and to particular cell populations is the challenge that is being addressed using a variety of viral and nonviral delivery systems which have distinct advantages and disadvantages. The nonviral vectors (synthetic vectors) are generally reputed to lack of efficiency while offering flexibility and safety. The suitability of any gene delivery system should always be matched with the clinical situation, the specific disease and the chosen therapeutic strategy.

Conceptually nucleic acid based therapies take two different approaches first the delivery of plasmid DNA or related constructs to express the gene of interest which result in the increased activity of the target and second the expression of oligomeric genetic material such as antisense oligonucleotide, siRNA or DNAzyme which result in reduction of target activity. None of the current vector systems is able to clearly satisfy the various diverse needs and it is therefore important to appreciate the strengths and weaknesses of synthetic vector systems in the appropriate therapeutic context [11]. Following are some strategies of gene delivery.

Barriers to gene delivery and pharmacokinetics of gene delivery in cells

In order that a DNA to transfect the cell it has to overcome barriers such as surviving the extracellular environment and entering the target cell type, cytosolic delivery from the endosomes, traversing to the nucleus from the cytoplasm and finally dissociation of the DNA from carriers for transcription.The extracellular environment presents the major hurdle the foreign would have to overcome. The nuclease capable of digesting unprotected DNA into fragments and eventually incapacitating its ability to express the encoded protein. One of the major approaches of the DNA overcoming degradative enzymes is to condense it into a compact form such that sites vulnerable to cleavages could be protected. This condensation is based on the electrostatic interactions between the anionic nucleic acid and the positive charges of the synthetic vector which complex and condense the NA into nanoparticles. Several poly cations such as Polyethylenimine (PEI), Poly-L-Lysine (PLL), cationic lipids, dendrimers, etc have been used for this purpose. Based on the synthetic vectors used the resulting particle were termed as polyplex, lipoplex and dendriplex respectively [11].

Synthetic vectors, regardless of the route of entry which could either be via receptor mediated endocytosis or by pinocytosis, they would be brought into the early endosomes, which would either fuse with other endocytic vesicles, more commonly late endosomes that exocytose internalized products. The sudden lowering of PH to 5 within the microenvironment of the late endosomes triggers the process of fusion between early and late endosomes. Late endosomes finally fuse with the lysosomes where degradation takes place due to acidic environment and degrading enzymes. Thus for a successful gene transfer to take place polyplexes have to escape from the endosomes before they fuse with lysosomes. To increase endoosmolytic property several strategies have been proposed one of them is to conjugate polycation with Mellitin, a major component of bee venom known to lyse cell membranes, a 25kDa PEI was conjugated with mellitin which showed augmented levels of endosomal release and nuclear transport [12].

In order that a delivered DNA to be functional it has to be transported into the nucleus where it could be transcribed into mRNA and ultimately translated into protein. This entry form cytoplasm to nucleus is governed by the nuclear membrane. The nuclear pore complex mediates the transport of molecules. This complex allows passive passage of small molecules but severely limits passage of larger molecules of more than 50kD across membrane [13,14].

Dissociation of carriers form plasmid is an essential process for the efficient transcription. To demonstrate these plasmids where micro injected into the nucleus and into the cytoplasm were compared, the plasmid injected into the nucleus gave rise to higher expression levels [15].

Pharmacokinetic considerations in optimization of intracellular trafficking

In order to optimize intracellular trafficking, it is necessary to balance various processes related to the rate-limiting intracellular barriers. The increased efficiency of one process may reduce that of others. For example tight condensation of pDNA, so as to produce small complex, enhances cellular uptake by endocytosis, but excess condensation inhibits transcription. A computer-assisted intracellular kinetic model with kinetic parameters (i.e. first order rate constant: time-1) determined using quantitative data is a useful tool to analyze, simulate and optimize transgene expression. An integrated kinetic model for cellular uptake, endosomal release, nuclear binding, nuclear translocation, dissociation, and protein synthesis with firstorder mass action kinetics was proposed by varga et al. 2001and demonstrated the utility of kinetic modeling for optimization [16].

Physical chemistry of DNA carrier complexes

The study of condensation of giant DNA molecules has been studied actively not only in order to develop non-viral gene therapies but also to understand self- regulation of genetic activity in living cells [17]. Extensive studies on the drastic change involving the confirmation of DNA known as DNA condensation have been carried due to that fact that DNA molecules in viral caspids, bacterial nucleoids, and nuclei of eukaryotes occupy a volume 10-4 to 10-6 times less then when free in aqueous solution [18]. Due to the limitations in available experimental approaches to analyze DNA condensation, such as light scattering, sedimentation, viscometry, linear dichroism, circular dichroism, and UV spectrometry, most studies dealing with DNA condensation have not clearly distinguished between transitions occurring in individual DNA molecules and those involving aggregation/ precipitation of numbers of DNA molecules [19]. The important aspect in relation to the manner of transition is the morphological changes in the compact state. As illustrated in Figure 1, when the transition is diffuse or continuous, the final compact state exhibits a spherical morphology with liquid drop like properties, whereas when the transition is discrete, an ordered packed state, generating toroid and rod morphologies, results [20]. DNA is a highly charged polymer with rather condensed arrays of highly acidic phosphate groups. Small counter – cations, such as sodium and potassium in physiological conditions are present at concentrations above mM. Thus it is important to consider the degree of dissociation of the sodium or potassium salt of the phosphate moieties along DNA chain [18]. According to the counter ion condensation theory, about 70% of the negative charge of DNA is neutralized because of the condensation of the counter ion. When a DNA molecule is tightly packed, almost all of the negative charge should be neutralized, accompanied by the enhancement of counterion condensation. Thus the volume part of the compact DNA is fully neutralized whereas negative charge remains on the surface [21]. An interesting finding in this aspect is that multivalent cations such as spermidine and spermine induces the folding transitions of DNA and this phenomenon is inhibited by the monovalent ions. Thus at higher salt concentrations, larger amounts of multivalent cations are necessary to induce the compaction. In some cases like folding transition induced in crowding environment, the presence of salt is a necessary condition, i.e., salt is a promoting factor for compaction [21].