Gene Delivery Vectors

The antitumor effect of gene therapy in prostate cancer is achieved when a therapeutic gene, under the control of a promoter, is delivered directly to a target cell by a vector. The ideal vector would be specific for prostate cancer cells and would have a high rate of transgene delivery. Furthermore, a vector should be inexpensive to produce and administer, nontoxic to the patient, and nonmutagenic. Two categories of vectors are used for human gene therapy, viral and nonviral; however, most prostate cancer clinical trials involve the use of viral vectors. The currently available vectors for gene delivery are listed in Table 1. Much research is underway to improve the specificity, transduction efficiency, and safety of gene therapy vectors.

The route of administration of the therapeutic agent depends by and large on the vector used. At the present time, most prostate cancer gene therapy trials involve the intralesional administration of the vector, which is very suitable because of the ability to visualize the prostate using transrectal ultrasound and its convenient transrectal or transperineal access. Ultimately, the desired route of administration is systemic delivery via intravenous injection of the vector. This approach would seek and destroy all foci of cancer, regardless of their location; however, limiting factors include vector half-life, hematological inactivation of the vector and the infection of nontarget organs. Recent improvements in prostate-specific promoter systems and viral targeting has allowed this approach to be used in human prostate cancer clinical trials.

2.1. Adenovirus

The most commonly used viral vector in human gene therapy clinical trials is adenovirus (Ad), a non-enveloped, icosahedral virus containing a double-stranded linear DNA genome, 36 kb in size. This nonintegrating virus has a wide safety profile and is advantageous over many vectors in part because of its ability to carry a large insert and infect any cell, regardless of its cell cycle status. In addition, its genome is easily manipulated in the laboratory, and it is readily produced in high titers with relatively minimal expense. Early generation adenoviral vectors induced strong innate and adaptive immune responses, thereby limiting their gene delivery potential (7). Furthermore, it has been

Structure Adenovirus

Fig. 1. Genome organization and structure of adenovirus. The transcription map of adenovirus (A) depicts the early transcription units with thin arrows, late transcripts in heavy arrows, and delayed mRNAs in italics. Encoded within the E3 region and under the control of the major late promoter is a late transcript, which includes adenoviral death protein (ADP). Map units are indicated by the hashes. Products of the late transcripts contribute to the structure of adenovirus (B). Proteins II, III, IIIa, IV, VI, VIII, and IX form the viral capsid. Proteins V, VII, X, and terminal protein (TP) associate with the adenoviral genome and form the viral core. Protease is responsible for the maturation of proteins IIIa, VI, VII, VIII, and TP. VA, virus associated; ITR, inverted terminal repeat.

Fig. 1. Genome organization and structure of adenovirus. The transcription map of adenovirus (A) depicts the early transcription units with thin arrows, late transcripts in heavy arrows, and delayed mRNAs in italics. Encoded within the E3 region and under the control of the major late promoter is a late transcript, which includes adenoviral death protein (ADP). Map units are indicated by the hashes. Products of the late transcripts contribute to the structure of adenovirus (B). Proteins II, III, IIIa, IV, VI, VIII, and IX form the viral capsid. Proteins V, VII, X, and terminal protein (TP) associate with the adenoviral genome and form the viral core. Protease is responsible for the maturation of proteins IIIa, VI, VII, VIII, and TP. VA, virus associated; ITR, inverted terminal repeat.

shown that nearly all humans have developed humoral immunity to Ad because of previous exposure; however, only 55% of the detected immunoglobulins are neutralizing antibodies (8). Recently, adenoviral vectors have been modified to reduce their immunogenicity, reinstating hope in their clinical usefulness (9).

The adenoviral genome, as depicted in Fig. 1A, is flanked on both ends by short inverted terminal repeats (ITRs), which contain identical origins of replication. Near one end is a short packaging signal (y), which is required for proper association of the viral genome with the capsid proteins (10). Its transcription units are divided into two groups, early and late genes. The viral genome carries six early units (E1A, E1B, E2, E3 , E4 , and E5), two delayed early units (lVa2 and IX), and two late units, one that is processed into five messenger RNAs (mRNAs) (L1-L5) and one in the E3 region that is controlled by the major late promoter. In addition, either one or two copies of virus-associated (VA) genes are encoded and transcribed by RNA polymerase III. As shown in Fig. 1A, the transcription of adenoviral genes occurs from both strands and uses alternative splicing and multiple poly(A) sequences.

E1A is the first sequence transcribed in the adenoviral genome. Controlled by a constitutively active promoter, the E1A transcription unit encodes up to five polypeptides, of which only two, E1A 12S and E1A 13S, have known functions. E1A proteins transactivate the transcription of other adenoviral genes (11). Expression of E1A is critical to the virus. In fact, deletion of the E1 region of the genome results in a replication-deficient Ad (12). E1A proteins also stimulate viral DNA synthesis by preventing Gi arrest and advancing the host cell into S-phase. This is accomplished by binding the tumor suppressor retino blastoma (pRB) and releasing the associated transcription factor E2F (13), antagonizing the cyclin-dependent kinase inhibitory protein, p27kip1 (14), or inhibiting the transactivation of p53 by p300/CBP (15). Furthermore, E1A proteins induce apoptosis of infected cells through p53-dependent and p53-independent pathways (15), both of which are blocked through the action of E1B proteins (16). Acting in concert, E1B and E4 proteins shut down host cell protein synthesis by blocking the cytoplasmic accumulation of cellular mRNAs, while stabilizing and exporting viral mRNAs from the nucleus to the cytoplasm (17,18). Although Ad is not responsible for human malignancies, the products of the E1A, E1B, and E4 genes transform cells in vitro (19); however, Ad5, the adenoviral serotype used in prostate cancer gene therapy belongs to a non-oncogenic subgroup. Viral DNA synthesis occurs as E2 gene products accumulate. The E2 region encodes a DNA polymerase that is essential for viral DNA replication (20) and the terminal protein which is covalently bound to the 5' ends of the viral chromosome and serves as a primer for DNA synthesis (21). The expression of E3 is not essential for viral replication; however, it protects virally infected cells from lysis by cytotoxic T-lymphocytes (CTLs) by downregulating the expression of major histocompatibility complex (MHC) class I antigen (22) and Fas receptor (23) on the infected cell surface. Further protection from the body's antiviral defense system is provided by VA RNA, which forms a hairpin-loop structure and inhibits the activation of interferon-induced RNA-dependent protein kinase (24).

Late gene products include 10 structural proteins, of which, 7 (II, III, Ilia, IV, VI, VIII, and IX) form the capsid and three (V, VII, and |) are involved with the DNA-containing core. Figure 1B depicts the structure of the Ad particle. The most abundant protein on the capsid surface is the trim-eric hexon (II), whose assembly requires the assistance of the L4 100-kDa scaffold protein (25). Neutralizing antibodies to the capsid are formed against surface loops of the hexon structure. Polypep-tides IIIa, VI, VIII, and IX stabilize the hexon capsid structure and form a bridge with the adjacent core proteins. The penton base (III) forms at the 12 vertices of the capsid structure, with a trimeric fiber and distal knob (IV) projecting from each base. Together, polypeptides III and IV form the penton complex and mediate host cell binding. Initially, the knob domain of polypeptide IV binds to its cellular receptor, the coxsackie and Ad receptor (CAR) (26), followed by binding of an arg-gly-asp (RGD) motif in the penton to avp3 and avp5 integrins on the cell surface, stimulating internalization (27). The E3 region encodes one late protein called Ad death protein, which is controlled by the major late promoter and is responsible for cell lysis and release of viral progeny (28). Recently, vectors have been constructed that overexpress Ad death protein, leading to enhanced viral spread and oncolysis (29).

Although prostate cancer cells upregulate CAR expression (30), tumors vary in adenoviral susceptibility. For this reason, efforts have been made to retarget the virus, thereby enhancing its ability to infect prostate cells. Furthermore, the detargeting of Ad has narrowed its expansive tropism, allowing for safer systemic delivery without infection of nontarget tissues, such as liver and respiratory epithelium. Early studies in adenoviral retargeting used bispecific antibodies that crosslinked the virus to alternative cellular receptors (31,32). This has been applied to prostate cancer recently, with a bifunctional antibody to the adenoviral fiber knob and prostate-specific membrane antigen (PSMA) (33). Perhaps a more clinically feasible approach to prostate-specific retargeting of Ad is the genetic modification of the fiber knob. Cell-binding peptides have been displayed on the carboxy terminus of the fiber knob (34); however, this approach is unfavorable because of size constraints and structural hindrance. Larger peptides can be incorporated into the HI loop of the fiber knob without structural consequences (35). Lupold et al. identified two candidates for prostate-targeting peptides by phage display library that bind to PSMA (36); however, there exist no reports of its use for the transduc-tional targeting of Ad. A final approach to the detargeting of Ad was devised by Shayakhmetov et al., in which the Ad5 fiber was replaced by the short-shafted Ad35 fiber (37). This chimeric Ad5/35 uses CD46 as its cellular receptor (38), which is upregulated on the surface of many cancer cells. Most importantly, systemic administration of these vectors does not result in hepatic infection, and Ad5/35 has demonstrated prostate cancer tropism (39). In combination with transcriptional targeting, the transductional detargeting and retargeting of adenoviral vectors will increase the safety and efficacy of systemically delivered molecular therapies for prostate cancer.

2.1.2. Adeno-Associated Virus

Adeno-associated virus (AAV), a member of the parvovirus family, is a small single-stranded DNA virus that is dependent on a helper virus, such as Ad, for replication. AAV is an attractive vector for gene therapy because it elicits nearly no immune response, is known to cause no disease in humans, and integrates stably and site specifically in a region on chromosome 19 (40). The genome consists of two ITRs, which encode a packaging signal and the origin of replication, and two genes, rep and cap. Cap encodes the viral capsid proteins whereas rep encodes four products of alternative splicing, Rep40, Rep52, Rep68, and Rep78, of which only Rep68 or Rep78 are necessary for replication and site-specific integration (41). Recombinant AAV vectors are made by deleting rep and cap and inserting approx 4.7 kb of therapeutic DNA; however, it has been observed that deletion of rep results in nonspecific integration of the virus (42). Nonetheless, the majority of AAV vectors used today are rep-deleted. Recombinant AAV is produced in HEK293 cells by co-transfecting a plasmid containing the therapeutic DNA cloned between two ITRs and a plasmid containing the rep and cap genes. Subsequently, the cells are infected with Ad or transfected with a plasmid containing the adenoviral genes required for AAV replication (43). Recently, methods have been devised to produce higher AAV titers, necessary for large-scale production for gene therapy applications (44). After infection, the rate-limiting step in gene expression seems to be the synthesis of the second strand (45). This has been overcome by the recent development of self-complementary, double-stranded AAV vectors that carry half the insert size of the single-stranded virus (46). Similar to Ad, peptides can be inserted into the AAV capsid to retarget the vector specifically to receptors on the surface of prostate cancer cells (47). To achieve site-specific integration of a transgene with the targeting capability of an adenoviral vector, Recchia et al. developed a hybrid Ad/AAV vector that carries a drug-inducible rep expression cassette and a transgene cassette flanked by two AAV ITRs. Site-specific integration of the ITR-flanked transgene cassette was observed in a rep-dependent fashion (48).

Was this article helpful?

0 0
How To Bolster Your Immune System

How To Bolster Your Immune System

All Natural Immune Boosters Proven To Fight Infection, Disease And More. Discover A Natural, Safe Effective Way To Boost Your Immune System Using Ingredients From Your Kitchen Cupboard. The only common sense, no holds barred guide to hit the market today no gimmicks, no pills, just old fashioned common sense remedies to cure colds, influenza, viral infections and more.

Get My Free Audio Book


Post a comment