What is the method of gene transfer?

SUMMARY

Gene transfer has played a significant role in understanding the biological activity of cytokines, the role of cytokines in disease and allowed for the development of clinical approaches for treating a wide variety of diseases. Gene transfer of cytokines to cells in culture or in vivo can be accomplished by a variety of gene transfer vectors, including both viral- and non-viral- based vectors. For specific gene transfer applications’ certain vectors have their own advantages and disadvantages. The majority of phase I clinical trials using cytokine gene transfer have not shown significant efficacy. However, it is likely that as we learn more about the biology of cytokines, especially how multiple cytokines work synergistically to regulate the immune response, it is likely that cytokine gene therapy will play a significant role in the treatment of many human diseases.

Abstract

Gene transfer can be useful for tissue engineering by modifying cells directly or providing a favorable growth environment for the engineered tissue. Several ex vivo or in vivo strategies have evolved to enhance targeting of gene transfer vectors, regulation of gene expression, and DNA editing. Gene therapy can be combined with stem-cell strategies to aid in controlling growth and circumvent rejection. There are still challenges for using gene transfer for tissue engineering, but the technology is sufficiently advanced for rapid progress, in particular as gene therapy has entered the clinical realm as therapy for genetic and nongenetic diseases.

Abstract

Gene transfer is an increasingly utilized approach for research and clinical applications involving the central nervous system (CNS). Vectors for gene transfer can be as simple as an unmodified plasmid, but more commonly involve complex modifications to viruses to make them suitable gene delivery vehicles. This chapter will explain how tools for CNS gene transfer have been derived from naturally occurring viruses. The current capabilities of plasmid, retroviral, adeno-associated virus, adenovirus, and herpes simplex virus vectors for CNS gene delivery will be described. These include both focal and global CNS gene transfer strategies, with short- or long-term gene expression. As is described in this chapter, an important aspect of any vector is the cis-acting regulatory elements incorporated into the vector genome that control when, where, and how the transgene is expressed.

Abstract

Gene transfer is a potentially powerful approach to stem cell biology and tissue engineering, by engaging and altering the software of cellular behavior. While drug or chemical-based therapeutics can certainly modify cellular behavior, no method matches the potential of gene transfer for precise manipulation of cellular programming.

The target tissues for craniofacial gene transfer therapy discussed in this chapter bear great similarity to those tissues where the greatest progress in clinical gene therapy has already been demonstrated (e.g., eye, muscle, skin, and bone), underscoring the potential power of gene transfer in dental and craniofacial therapeutics.

Several proof-of-concept ex vivo and in vivo gene transfer strategies have been employed in pre-clinical studies, with numerous other prospective in vivo gene transfer approaches for the management of pathologic processes involving the oral and maxillofacial region at earlier stages of development. However, recognizing the many necessary safety and regulatory issues involved in the development of any gene transfer study, it is not surprising that the few successful stage 3 gene transfer trials to date have largely focused on the treatment of rare single gene Mendelian-inherited diseases. Nevertheless, as illustrated in this chapter, the potential conditions and approaches to gene therapy for conditions involving the head and neck complex are wide-ranging.

GENE TRANSFER TO CEREBRAL BLOOD VESSELS

Gene transfer is a useful tool for study of vascular biology, and despite great obstacles, the application of such therapy to cerebrovascular disease has considerable potential.150151 The goal is to introduce complementary DNA (cDNA) into a cerebral artery or perivascular tissue and to stimulate production of a protein that favorably modulates vascular growth or function. Theoretically, this could be accomplished with the use of naked DNA, but the approach provides very inefficient gene transfer. Thus, more efficient vectors have been developed, including viral vectors (adenovirus, retrovirus, adeno-associated virus, and herpesvirus), complexes of DNA-cationic lipids, and viral conjugate vectors (liposomes with a viral coat).150 Each of these vectors has specific advantages (related to efficiency, safety, trophism, and ease of preparation), and each has important limitations.151 An "optimal" vector is not yet clearly identified.

Gene transfer can potentially be "targeted" to vascular tissues, such as adventitia or endothelium, or to specific receptors on endothelium.152 Transgene expression may be driven by tissue-specific promoters, and regulated expression will be valuable. In addition, gene transfer can be used to express antisense oligonucleotides that inhibit expression of selected genes. For example, an antisense construct for astrocyte glial fibrillary acidic protein has been used to inhibit gliosis in astrocytes.153

It is now possible to accomplish gene transfer to cerebral vessels. Several studies demonstrate expression of functional proteins after gene transfer to carotid arteries and cerebral blood vessels.154155156157158159160 Reporter genes, such as β-galactosidase, were used in some studies to demonstrate the feasibility of gene transfer.154161 In later studies proteins that modify vascular function, such as eNOS, have also been expressed in blood vessels through the use of gene transfer.162163164165166

Early approaches to producing gene transfer to blood vessels in vivo involved intraluminal methods that required stopping blood flow to allow uptake of vectors.155 To avoid ischemia reperfusion after interruption of blood flow, alternative methods have been developed to produce expression of transgene products in blood vessels. Promising methods are intracerebroventricular and intracisternal (cisterna magna) injection of viral vectors into cerebrospinal fluid.161167168169

Gene transfer by intravenous injection of vectors results in high levels of transgene expression in liver and lung, but not in efficient expression in vessels. Protection against stroke has been reported, however, after gene transfer accomplished by intravenous gene transfer of atrial natriuretic peptide or kallikrein.170171 The mechanism of neural protection is not clear and may have resulted from reduction in blood pressure or response to circulating transgene products rather than from local effects of gene transfer to cerebral vessels. The studies nevertheless suggest that intravenous administration of vectors expressing some genes has potential for treatment or prevention of stroke.

One strategy for treatment of stroke assumes that progressive tissue injury, primarily in ischemic penumbral regions, may be inhibited. Thus, gene transfer may be useful in providing continuous local production of a therapeutic gene product to the penumbral region for several days after ischemia (Fig. 37-4).

Preliminary studies have been performed to evaluate the therapeutic potential of gene transfer to reduce ischemic brain injury. Adenovirus-mediated gene transfer of the lacZ gene, by direct injection into brain, produces high levels of expression of β-galactosidase in brain.172 Thus, gene transfer can be accomplished in the brain after ischemia. This study is important in addressing the possibility that cerebral ischemia might impair synthesis of proteins after gene transfer.

Other studies report that intracerebroventricular injection of adenoviral vectors over-expressed an interleukin-1 receptor antagonist before ischemia and significantly reduced infarct size.173 Neuroprotection against ischemic infarction has been reported after gene transfer of glial cell line-derived neurotropic factor174 and atrial natriuretic peptide.170 Many gene products have potential for therapeutic roles in protection against ischemic brain damage.

Another approach is to perform gene transfer to cells ex vivo and to surgically implant the transfected cells into blood vessels. In one study, tissue-type plasminogen activator protein (tPA)175 was measurable in carotid arteries after implantation of cells exposed to adenoviral-mediated gene transfer ex vivo. Because tPA is used to treat ischemic stroke, the potential for gene therapy to express this substance is provocative.

Several studies suggest a future for gene therapy after SAH. Gene transfer to cerebral vessels can be accomplished after SAH,176 suggesting that formation of a thrombus does not preclude intervention. Gene transfer of calcitonin gene-related peptide (CGRP), an extremely potent dilator of cerebral vessels, reduces vasospasm after SAH in rabbits.177178 Other laboratories have reported that gene transfer of eNOS attenuates vasospasm and improves vasodilator responses after SAH.179180 Thus, several interventions based on gene therapy may have therapeutic potential after embolic stroke and SAH.

Gene transfer might also be used to stimulate growth of collateral vessels in the presence of ischemia. This approach appears feasible in the peripheral circulation. Gene therapy might be used to treat brain tumors through inhibition of vascular proliferation, which thus would produce ischemia in the tumor.

Gene therapy might be useful for treatment of extracranial vascular disease, such as in the carotid or vertebral artery. Gene transfer approaches have been used to interrupt the cell cycle and thereby inhibit proliferation of vascular muscle. A goal of gene therapy might be to transfect an intracranial or extracranial artery after angioplasty with a gene that inhibits proliferation or remodeling. The problems related to inflammation and transient expression of transgenes provide major obstacles for the use of gene therapy at present.

Sperm-Mediated Gene Transfer

SMGT is based on the intrinsic ability of sperm to bind and internalize exogenous DNA and to transfer it into the egg during fertilization. The basic steps of SMGT in pigs are the collection of sperm, the incubation of sperm with exogenous DNA, and the artificial insemination of gilts with DNA-loaded sperm. Various modifications for the different steps of the method were described. Establishment and reproducibility of SMGT between laboratories for routine use are still missing since it was first described in mice. Therefore, only few transgenic lines were produced by this method to date.

A crucial point of the technique is the selection of suitable sperm donor animals.23 The efficiency of SMGT of the human CD55 (decay accelerating factor, DAF) transgene in pigs was reported to be up to 57%, i.e. 53 out of 93 founder pigs were transgenic. Most of the CD55 transgenic pigs expressed the transgene in a stable manner (64%) and transgenic founders transmitted the human CD55 expression vector to their progeny.24 Thus, the method has the potential for high efficiency by using a simple technique with a low number of animals involved and decreased manipulation of the embryos.

Improvement of SMGT in pigs was studied by using a mouse monoclonal antibody capable of binding to sperm. This linker protein bound to a surface antigen on sperm and facilitated the binding of exogenous DNA to sperm via ionic interaction. Using a transgene consisting of the secreted human alkaline phosphatase under the control of the SV40 early promoter and enhancer, transgenic founder pigs were produced by insemination. They showed the expression of the transgenic protein and germline transmission of the transgene to the offspring.25

The intracytoplasmic sperm injection (ICSI)-mediated gene transfer is a technique related to SMGT and has been successfully carried out for producing transgenic pigs. Pretreatment of the sperm heads by freeze thawing before incubation with exogenous DNA seemed to be useful in ICSI-mediated gene transfer.26

Attempts to increase the efficiency of SMGT resulted in the production of transgenic pigs using ICSI–SMGT in combination with recombinase RecA. After having optimized the experimental conditions for sperm function and in vitro production of transgenic embryos by ICSI in combination with RecA, transgenic pigs were produced, however, without visibly expressing green fluorescent protein (GFP), which was used as reporter gene.27

I INTRODUCTION

Gene transfer is a therapeutic strategy using genetic information, usually in the form of DNA, to modify the phenotype of cells. Gene therapy strategies can be useful for tissue engineering by modifying cells directly or by providing a favorable growth environment for the engineered tissue. To accomplish this, cells are modified genetically ex vivo or in vivo using gene transfer vectors that mediate the transfer of therapeutic DNA into the nucleus, where it is transcribed in parallel with genomic DNA. A variety of nonviral and viral gene therapy vectors have been developed, including plasmids, plasmids combined with liposomes, adenovirus, adeno-associated virus, retrovirus, and lentivirus. A number of strategies have evolved to enhance the targeting of gene transfer vectors by genetic or chemical modification of the surface of the vector. Gene expression directed by the transferred gene can be regulated by including inducible promoters, tissue-specific promoters, and trans-splicing. Although still in the early stage of development, there is significant potential to combine gene therapy with stem cell strategies to aid in controlling cell growth and to circumvent immune rejection. There are still challenges to using gene transfer for tissue engineering, but the technology of gene transfer is sufficiently advanced that rapid progress should be made in the near future.

Gene therapy uses the transfer of genetic information to modify a phenotype for therapeutic purposes (Verma and Weitzman, 2005). The application of gene transfer to tissue engineering has myriad possibilities, including the transient or permanent genetic modification of the engineered tissue to produce proteins for internal, local, or systemic use, helping to protect the engineered tissue, and providing stimuli for the engineered tissue to grow and/or differentiate. To provide a background for the application of gene transfer to tissue engineering, this chapter reviews the general strategies of gene therapy, details the gene transfer vectors used to achieve these goals, and discusses the strategies being used to improve gene transfer by modifying the vectors to provide cell-specific targeting and by regulating the expression of the targeted gene. The applications combining gene therapy with stem cell therapy are reviewed. Our overall goal is to provide a state-of-the-art review of the technology of gene therapy, including the challenges to making gene therapy for tissue engineering a reality. For details regarding the applications of gene therapy to specific organs and clinical disorders, several reviews are available (Anderson, 1998; Crystal, 1995; Kaji and Leiden, 2001; Lundstrom, 2003; O'Connor and Crystal, 2006; Thomas et al., 2003; Verma and Weitzman, 2005).

14.4.6 Alternative Methods for Cardiac Gene Transfer

Gene transfer of peptides with paracrine activities that may benefit the heart is an alternative to cardiac-targeted gene transfer and may be applicable for CHF and other cardiovascular diseases. A prerequisite for this approach is the selection of a transgene that has cardiac effects after being released to the circulation from a distant site. We have tested this concept using skeletal muscle injection of AAV5 encoding IGF-I under tetracycline regulation (AAV5.IGFI-tet).93 In this study, AAV5.IGFI-tet was injected in the anterior tibialis muscle in rats with severe CHF induced by MI. Activation of IGF-I expression by addition of doxycycline to the drinking water increased serum IGF-I levels and improved function of the failing heart. This new approach enables transgene expression at a remote site and circumvents the problem of attaining high yield cardiac gene transfer. As mentioned previously, IV delivery may be superior to intramuscular delivery.

3.2 Efficiency

Adenoviral gene transfer was assessed by histochemistry (X-gal staining for LacZ expression) in the whole heart after 2-3 days of infection. As shown in Fig. 2, X-gal staining was homogeneously distributed throughout the multiple cross sections of the heart following gene delivery by Protocol 1. Figure 3 illustrates the level of gene transfer by Protocol 2. The expression was low and heterogeneous. Cells were isolated from stained hearts from both protocols to determine the efficiency of gene transfer, as the number of cells stained blue for exogenous gene expression relative to total cell counts. Efficiency was 58 ± 11% for Protocol 1 and <5% for Protocol 2. The low level of gene transfer for Protocol 2 is consistent with earlier reports [3,10,11].

Gene transfer of drug-resistance (CTX-R) genes can be utilized to protect the hematopoietic system from the toxicity of anticancer chemotherapy, and this concept recently has been proven for O6-methylguanine methyltransferase in the context of temozolomide therapy of glioblastoma patients. Given its protection capacity against such relevant drugs as arabinoside cytosine, gemcitabine, or 5-azacytidin and the highly hematopoiesis-specific toxicity profile of several of these agents, cytidine deaminase (CDD) represents another particularly interesting candidate CTX-R gene. Recently, our group established the myeloprotective capacity of CDD gene transfer in a number of murine transplant studies. Although these studies also have highlighted the problems and risks still associated with CDD gene transfer, such as lymphotoxicity, lack of long-term in vivo selection, insertional mutagenesis, or inadvertent transduction of malignant cells, these may be overcome by recent developments in the field, such as safety-improved self-inactivating vector constructs, regulated transgene expression systems, or novel CTX-R genes.