How can a gene be delivered to the cell?

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.

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

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.

D. AADC

Gene transfer of AADC to increase dopamine levels has been investigated in both rat and primate PD models (Leff et al., 1999; Bankiewicz et al., 2000; Sanchez-Pernaute et al., 2001). Transduction of striatal tissue with AAV-AADC combined with an exogenous dose of l-dopa in dopaminergically denervated animals resulted in the phenotypic correction of motor deficits. This was proposed to be due to increased decarboxylation of l-dopa to dopamine as a result of increased AADC levels, and suggests that gene transfer of AADC by itself may be beneficial in PD treatment by reducing the dose of l-dopa required to give relief from motor abnormalities, thereby avoiding or delaying development of side effects from l-dopa administration.

Gene transfer to control uncontrolled stem cell growth

Gene transfer strategies developed to control the growth of cells for cancer gene therapy applications can be applied to controlling the unwanted growth of stem cells, in particular, the risk of the development of teratomas with embryonic stem [183]. Some of the suicide gene transfer strategies, including the prodrug strategies using herpes simplex thymidine kinase (HSV-TK) (ganciclovir) and cytosine-deaminase (5-fluorocytosine) that lead to activation of cell death following administration of the prodrug may prove useful. Other suicide genes used with regulatable gene expression systems (as outlined above, Table 34.4) may be useful for controlling potential malignant stem cell growth, as long as there is no ’leak’ of baseline gene expression of the suicide gene.

Polynucleotides

Gene delivery from the surfaces of tissue scaffolds represents a new approach to manipulating the local environment of cells [111]. Gene therapy approaches can be employed to increase the expression of tissue inductive factors or block the expression of factors that would inhibit tissue formation [112]. A biomaterial can enhance gene transfer by localized expression of the genetic material and by protecting the genetic material against degradation by nucleases and proteases. Sustained delivery of DNA from a polymer matrix may transfect large numbers of cells at a localized site and lead to the production of a therapeutic protein that could enhance tissue development. The work by Yao [113] serves as a specific example for this approach: Yao investigated the potential of chitosan/collagen scaffolds with pEGFP-TGFβ1 as a gene vector candidate in cartilage tissue engineering.

C Gene Therapy in Hemophilia A Dogs

Gene transfer remains an important and active area of research and clinical investigation. All three initial human trials for gene therapy hemophilia A were stopped due to low-level expression or vector toxicity.298–300 Two of these three trials were tested in the Chapel Hill hemophilia A dogs and their outcomes were presaged by the results of these preclinical trials.301,302 Fortunately, no inhibitor to FVIII was detectable after gene transfer in these patients. Several gene therapy strategies have been tried using retroviral, adenoviral,303,304,82 lentiviral,305 and AAV vectors.306,307 Currently, novel serotypes of AAV are showing continued progress with expression up to 20% of normal, levels that would prevent spontaneous bleeding in hemophilia A patients.98 Importantly, a strategy that coadministered the proteasome inhibitor bortezomib with AAV vectors expressing the relatively oversized FVIII transgene have documented multi-year expression.102 This breakthrough has the potential to expand the applicability of AAV vectors for gene therapy in general but especially for large cDNAs such as FVIII. Most importantly, all animals undergoing gene transfer continue to be monitored for the long-term (i.e., years) safety and efficacy of all of these new methods. An unexpected but a highly desirable outcome in these gene transfer studies has been transgene expression over 10 years albeit at low levels but without detectible toxicity.168,308 These observations are particularly important when considering the occurrence of hepatocellular carcinoma and angiosarcomas in mice with the lysosomal storage disease mucopolysaccharidosis type VII (MPSVII) after treatment with rAAV vectors.309,310 Insertional mutagenesis is probably the operative mechanism but other possibilities include overexpression of a human transgene in rodents and colony contamination by oncogenic viruses. To address this issue we are screening all AAV-treated Chapel Hill hemophilia A (or hemophilia B described below) dogs for evidence of tumor formation or other pathology at the sites of gene transfer. The availability of relevant animal models that survive for 10 years after gene therapy with a species-specific transgene has proven invaluable to assess toxicity over time.

1 In Vitro

Gene transfer in the laboratory setting, particularly the use of plasmid transfections (e.g., calcium phosphate precipitation and liposome-mediated electroporation) in vitro with cultured cells, is commonly used today to address a wide variety of important biological questions. The value in using viral vectors, especially ade-noviral vectors, for gene transfer in vitro is that these vectors are highly efficient in transferring genes. Thus, by using a relatively modest dose of an adenoviral vector an investigator can routinely and reproducibly transfer the gene of interest to 100% of the target cells. Alternatively, gene transfer to a lesser fraction of cells can be reproducibly attained if desired. This approach has been helpful for our studies examining the relationship between the expression of aquaporin-1, a water channel protein, and transepithelial fluid movement (Delporte et al., 1998; Hoque et al., 2000).

In our experience, use of adenoviral vectors for gene transfer can offer a significant practical advantage to the investigator. Plasmid transfection techniques are inefficient and many experiments will ultimately require a subsequent selection step (e.g., antibiotic selections) in order to enrich (or make “uniform”) the cell population employed to test the biological question of interest. Cell selection methods are not necessary for obtaining uniform transgene expression if adenoviral vectors are used for gene transfer. Although it is true that to construct a first-generation adenoviral vector takes time, the methods for doing so have greatly improved (T. C. He et al., 1998). With the gene of interest in hand, it is possible to generate a new adenoviral vector in quantities suitable for cell culture use within 7–10 days’ time.

Background

Gene transfer has developed into one of the main experimental tools in neuroscience to explore and to understand the functions of various proteins. This technology is designed to manipulate gene expression in large populations of neural cells, in dissociated and organotypic slice cultures, and in vivo. Moreover, gene transfer is beginning to be used for therapeutic purposes. Recombinant viral vectors provide one of the most efficient methods of gene transfer in the CNS.

The two main routes to transfer genes to the CNS, the indirect (ex vivo) and direct (in vivo), have their advantages and disadvantages. The direct in vivo gene transfer, by recombinant viral vectors such as adenoviral vectors, adeno-associated viral vectors, and lentiviral vectors, entails transduction of nondividing resident host cells and modifying them genetically, with long-lasting and nonreversible expression of the transgene. In ex vivo gene transfer, the vector is first delivered to a cell line in vitro, and these cells are then transplanted into the brain. In this case, there is no integration of foreign DNA causing a genome dysregulation of the host cells. Further, the special case of encapsulated cell biodelivery (ECB) provides an additional safety aspect to the procedure, as it is possible to remove the cell-containing capsules in case of unwanted side effects. Both direct (in vivo) and indirect (ex vivo) gene transfers have been used to control epileptic seizures in animal models with variable success, depending on the targeted genes and cells.