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Tissue Engineering - John P. Fisher

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13-20

Tissue Engineering

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14

Gene Therapy

 

14.1

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

14-1

 

14.2

Nucleotides for Delivery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

14-2

 

 

DNA (deoxyribonucleic acid) RNA

14-2

 

14.3

Gene Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

 

 

Biological Delivery Methods Chemical Delivery Methods

 

 

 

Physical Delivery Methods

14-6

 

14.4

Intracellular Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

 

14.5

Cell and Tissue Targeting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

14-7

 

14.6

Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

14-8

J.M. Munson

 

In Vitro Ex Vivo In Vivo

14-9

14.7

Clinical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

W.T. Godbey

14.8

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

14-10

Tulane University

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

14-10

14.1 Introduction

Gene therapy is the delivery of genetic material into cells for the purpose of altering cellular function. This seemingly straightforward definition encompasses a variety of situations that can at times seem unrelated. The delivered genetic material can be composed of deoxyribonucleic acid (DNA) or RNA, or even involve proteins in some cases. The alteration in cellular function can be an increase or decrease in the amount of a native protein that is produced, or the production of a protein that is foreign. The delivery of the genetic material can occur directly, as is the case with microinjection, or involve carriers that interact with cell membranes or membrane-bound proteins as a part of cellular entry. Polynucleotides can be single or double stranded, and can code for a message, or not (as is the case for antisense gene delivery). Even the location of cells at the time of gene delivery is not restricted. Cells can be part of a living organism, can exist as a culture on a plate, or can be removed from an organism, transfected, and replaced into the same or a different organism at a later time.

Gene therapy came into being after the development of recombinant DNA technology and initially moved forward using viruses to target sites in vitro [1]. With the understanding of retroviruses and their possible uses as vectors for delivery, gene therapy progressed to applications involving mammalian organisms during the 1980s [1]. Since then, new techniques for gene delivery have been developed that utilize both viral and nonviral carriers. These techniques have been successful enough that proposed disease treatments have evolved to the clinical trial stage with encouraging success. Although many cellular processing mechanisms remain unclear and the search for the ideal gene delivery vector remains ongoing, the tools and methods for gene therapy have provided a strong base from which to build.

14-1

14-2

Tissue Engineering

14.2 Nucleotides for Delivery

14.2.1 DNA (deoxyribonucleic acid)

DNA is the building block of life and as such it remains a staple in the delivery of genetic material to the cell. There are many strategies as to how the DNA will interact with the existing genome and what is the best way to produce the desired effect.

14.2.1.1 Plasmids

A plasmid is a circular piece of DNA. Extrachromosomal plasmids can be replicated or transcribed independently of the rest of the DNA in the nucleus. This attribute makes plasmids an ideal tool for gene therapy. Plasmids can include a selectable marker for identification of transfectants/transductants, a multiple cloning site for ease of inserting/deleting additional nucleotide strands, the exon(s) of interest, and regulatory/binding elements such as promoters and enhancers. A common promoter used in gene therapy is the cytomegalovirus immediate early promoter (CMVie), used because of its strength in transcription initiation in nearly every mammalian cell. However, several other promoters have been investigated, such as the glucose-related protein promoter [2] and many cell-specific promoters. Enhancers include the simian virus 40 (SV40) enhancer as well as long terminal repeats (LTRs) [2]. The exons that are delivered may code for necessary cell-specific proteins, engineered protein polymers [3], or what has become a common objective in cancer cell research: suicide genes [4].

14.2.1.2 Nucleotide Decoys

DNA and RNA decoys are small oligonucleotide fragments that mimic the start sequences of potentially harmful genes. By doing so, the decoys can effectively trap the transcriptional and translational machinery, thereby causing a sharp decrease in mRNA or protein production. There has been success with this approach in human immunodeficiency virus (HIV) research. DNA and RNA decoys have been used to halt the function of REV proteins, which are responsible for making late transcripts of RNA and exporting them to the cytoplasm [5], and the TAT protein, which binds and activates the natural promoter for HIV-1 [6].

14.2.2 RNA

RNA also holds a valuable place in gene therapy because it can be used to alter cellular function. Two examples of RNA use in gene therapy are antisense RNA and small interfering RNA (siRNA). Antisense RNA provides functional alterations in cells by acting on host gene products post-transcriptionally. Antisense RNA functions by binding to cellular mRNA transcripts, which prevents ribosomal binding and therefore translation. Antisense RNAs have been investigated for their use in applications such as beta-globin gene inhibition as a possible treatment for sickle cell anemia [7], bcr/abl interference in myeloid leukemia research [8], and CXCR4 disruption for HIV-1 gene therapy [9], among others. The use of siRNA can interfere directly with genomic DNA transcription. First described by Elbashir et al. [10] in 2001, siRNAs have very high specificity that can be used to target a single-nucleotide polymorphism (SNP) on a single mutant allele [11,12]. The result would be to silence the mutant allele while permitting continued transcription of the wild type gene. This technology has been used in vivo to successfully target spinocerebellar ataxia type 3 and frontotemporal dementia [13].

14.3 Gene Delivery

Simply administering naked genetic material in the vicinity of cell exteriors is usually not sufficient to bring about the desired cellular response at adequate levels. Carriers have been employed to aid in the transfer of genetic material from the cell exterior to the cytoplasm or nucleus. There is a wide variety of carriers available, each with links back to one of the main branches of natural science: biology, chemistry,

Gene Therapy

 

14-3

 

TABLE 14.1 Examples of Virus Families and Members Used for

 

Gene Delivery

 

 

 

 

 

 

 

 

 

Family

Example

References

 

Adenoviridae

Adenovirus

[14]

 

 

Baculoviridae

Baculovirus

[15]

 

 

Herpesviridae

Pseudorabies virus

[16]

 

 

 

Simplex virus

[17]

 

 

Parvoviridae

Adeno-associated

[18]

 

 

 

Parvovirus

[19]

 

 

Poxviridae

Vaccinia

[20]

 

 

 

Yaba monkey tumor virus

[21]

 

 

Retroviridae

Human immunodeficiency virus 1 and 2

[22,23]

 

 

 

Lentivirus

[24]

 

 

 

Murine leukemia virus

[25]

 

 

 

Retrovirus

[26]

 

 

 

 

 

 

and physics. It is possible for hybrid systems that combine two or more basic technologies to exist, but the individual classifications will be discussed separately for clarity.

14.3.1 Biological Delivery Methods

Because they are a product of millions of years of biologic evolution, viruses are included here as the biological forms of gene delivery in spite of the fact that viruses are not technically alive (they do not respire). Viruses are the most efficient vectors for large-scale gene delivery. Some also offer permanent expression of delivered genes through the integration of genetic material into the transduced organism’s genome. Table 14.1 lists some of the major viral families and specific members that have been used in gene therapy. Many of the references in the table cite review articles of the specific virus classification for further reading.

Currently, herpes simplex virus (HSV) is being used to transduce cells in the central nervous system. By altering the genome of the virus, researchers have been able to decrease cytotoxicity and increase transfection efficiency [27]. HSV is also desirable because it is relatively large to allow packaging of larger plasmids, does not integrate its DNA into the host genome which avoids the deleterious effects that are possible with random integration, and is easily deliverable into the brain due to its ability to actively travel towards the nuclei of the neurons through the axons in the peripheral nervous system. HSV has also been applied to the transduction of skeletal muscle. Recent reports have described successful HSV-mediated gene transfer into skeletal muscle cells in vitro and in vivo and outlined possible applications for the delivery of large genes such as that coding for dystrophin [28].

In the liver, the virus AcM NPV (of the family baculoviridae) has yielded promising transfection efficiencies [29]. The YABA-like virus, which exhibits very similar attributes to the vaccinia virus but does not produce immune responses in the host, has successfully targeted and treated ovarian cancer in mice [30].

Adeno-associated virus (AAV) requires coinfection with a helper virus (typically adenovirus) to be productive. AAV offers an advantage to the gene therapist in that it has been reported to provide permanent expression of delivered genes. In utero experiments in mice and nonhuman primates indicate that recombinant AAVs can successfully alter the genome of the host organism to produce stable transductants [31]. Adeno-associated viruses are commonly used and successful in targeting the central nervous system [32]. Although repeated viral transduction is associated with inflammatory and immunological responses in the host, it has been reported that these responses can be reduced by carefully balancing recombinant AAV dosing with prudent timing [33].

Retroviruses offer good transduction efficiencies in vivo. Hallmarks of retroviral transduction are that RNA is delivered into cells as opposed to DNA, cDNA is made using viral reverse transcriptase, and this cDNA is introduced into the host genome via the protein integrase to produce stable transfectants. Immunodeficiency viruses are commonly investigated retroviruses, and include HIV and feline

14-4

Tissue Engineering

immunodeficiency virus (FIV). Investigations into retroviral genome alterations, such as self-inactivation via promoter deletion, are being conducted in an attempt to render HIV-1 a safe vector for gene transfer [34]. Use of FIV has shown efficient transduction of nondividing cells without many of the inherent risks associated with delivering recombinant HIV to humans [35]. Lentivirus, which belongs to the same family as HIV and FIV, has been developed to produce concurrent regulated multiple gene delivery upon transduction [36].

14.3.2 Chemical Delivery Methods

Many chemical methods of transfection have been engineered and utilized in the past 20 years. The use of synthetic gene delivery vehicles, such as polymers or cationic lipids, offers several advantages. Nonviral gene delivery is not restricted to plasmid DNA sizes based upon the ability to fit into a viral head of finite size, as is evidenced by successful delivery of a 2.3 Mb artificial human chromosome using poly(ethylenimine)(PEI) [37], and a 60 Mb artificial chromosome into Chinese hamster ovary cells using liposomes [38]. Although nonviral gene delivery usually results in transient gene expression, an advantage of this characteristic is that there is little or no risk of random integration into host genomes. Random integration poses a problem in that it can knock out a vital gene such as a housekeeping gene or a tumor suppressor gene. An additional advantage of nonviral gene delivery is the reduced threat of immune response in vivo versus repeated viral injections.

Several cationic polymers exist that exhibit strong transfection qualities. Many of these polymers are based on polypeptide chains containing multiple lysine, histidine, or arginine residues. Historically, poly(l-lysine) has been the most commonly used peptide transfection agent. However, several argininebased combinations of peptides have been formed into polyplexes of varying sizes and charge ratios which may yield better transfection results than the previously used polypeptide chains composed of a single amino acid [39]. Histidine–lysine polyplexes have also produced good transfection results that positively correlated with the amount of histidine in the complex [40]. There are also additions that can be made to these complexes in order to increase transfection efficiency and lower cytotoxic effects. These include ligands such as nerve growth factor, and hydrophilic polymers such as polyethylene glycol (PEG), which has been shown to increase transfection efficiency and lower cytotoxicity of poly(l-lysine) both in vitro and in vivo [41].

PEI is a highly cationic polymer that is available in both linear and branched forms. Each form of the polymer has a repeating [–CH2–CH2–N<] structure, with each nitrogen taking the form of a primary (end group), secondary (middle of a straight chain), or tertiary (branch point) amine (Figure 14.1). The groups attached to secondary and tertiary amines are typically additional [–CH2–CH2–N<], although terminal amines can be modified through the attachment of moieties for targeting or degradation purposes. Because of the large number of amines present in PEI, many of which carry a positive charge at physiological pH, DNA and RNA easily bind with the polymer via electrostatic interactions to form stable complexes. The transfection efficiency of branched PEI correlates with its molecular weight, with weight-average molecular weights of approximately 25,000 Da working best [42,43]. PEGylation of PEI polymers can, as was also the case for poly(l-lysine), increase transfection efficiency; this has been indicated in delivery of complexes to the central nervous system in vivo [44]. PEI has also been successfully used in in vitro transfections of rat endothelia and chicken embryonic neurons as well as in vivo in mice [45]. Conjugates of PEI are also being developed, such as silica microspheres coated with the polymer–DNA complexes. These conjugates have been used successfully for in vitro transfection, and offer the potential for relatively simple covalent surface modifications [46].

Dendrimers are highly branched polymers built around a single atom or molecule. Poly(amidoamine) (PAMAM) dendrimers are often used for gene delivery because of their high nitrogen content which aids in DNA binding and condensation. With a central atom of nitrogen, these polymers are built by the addition of amine-containing molecules, such as [CH2–CH2–NH2] (Figure 14.1). The result is a maximally branched molecule of known molecular weight; a sort of controlled version of PEI. PAMAM dendrimers, sometimes referred to as starburst dendrimers, have shown good transfection efficiencies in

Gene Therapy

14-5

(a)

H3C-CH2-NH CH2-CH2-NH CH2-CH2-NH2 n

(b)

 

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FIGURE 14.1 Three polymers based upon the [–CH2–CH2–N<] basic unit. Amines are shown in the uncharged state for clarity. (a) Linear PEI; (b) An example of a branched PEI. The polymerization permits 2amines and one cyclization via a back biting reaction. The polymer is somewhat irregular; (c) Third generation PAMAM dendrimer. All amines are tertiary amines (except for chain termini).

mammalian cells with little or no cytotoxicity in vitro [47]. A current application of this dendrimer is its coupling with an Epstein–Barr virus-based plasmid for suicide gene therapy in cancer cells [48].

Chitosan is another polymer that has been well characterized for use in transfection [49]. This natural nontoxic polysaccharide lends DNAase-resistance to its cargo while condensing the DNA to form stronger complexes [50]. The efficiency of chitosan is thought to rely upon its ability to swell and burst endolysosomes, which allows the delivered DNA to continue its path to the nucleus [51].

Alginate, a polycationic polysaccharide that can be used in the form of a hydrogel, has been used alone for transfection [52], as well as in combination with poly(l-lysine) complexes to form an oligonucleotide “sponge” [53]. Through slow degradation, these gels were shown to release embedded oligonucleotides over time. Possible applications of these gels to deliver antisense RNA are currently being pursued [53].

Microgels are similar to the alginate complex just mentioned. A thermosensitive microgel, composed of poly[ethylene glycol-(d, l-lactic acid-co-glycol acid)-ethylene glycol] (PEG–PLGA–PEG) triblock copolymers, has been created to have the property of being a liquid at room temperature but solidifying at 37C. Once solidified, the gel slowly degrades over the course of 30 days, releasing its component oligonucleotides to the surrounding cells. Possible applications of this gel system to wound healing are being pursued [54].

Lipids, particularly cationic lipids, have been used for gene delivery in a process termed lipofection. Several lipofection agents have been used for successful transfection of cells both in vitro and in vivo. Because of the aqueous environment inside cells and tissues, the hydrophobic tails of the lipids will coalesce to form hollow micelles, the interiors of which can contain oligonucleotides for cellular delivery. The combination of more than one hydrophobic entity can often yield higher transfection efficiencies than using a single type of lipid. In primary cortical neurons, a combination of N -[1-(2,3-dioleoyloxy)propyl]-N ,N ,N -trimethyl- ammonium methylsulfate and cholesterol (DOTAP : Chol) shows high transfection efficiency [55]. Using intraveneous administration of N -[1-(2,3-dioleoyloxy)propyl]-N ,N ,N -trimethylammonium chloride– Tween 80 (DOTMA-Tween 80) complexes with high DOTMA : DNA ratios, transient transfection to several organs has also been accomplished [56]. The use of helper lipids such as cholesterol or dioleoyl phosphatidylethanolamine (DOPE) can increase transfection efficiency significantly through endosomal membrane destabilization [57].