Ugrás a tartalomhoz

Molecular therapies

Dr. Zoltán Balajthy, Dr. János Aradi, Dr. Zoltán Balajthy, Dr. Éva Csősz, Dr. Beáta Scholtz, Dr. István Szatmári, Dr. József Tőzsér, Dr. Tamás Varga (2011)

University of Debrecen

3.2. Types of gene transfer, vectors for gene therapy

3.2. Types of gene transfer, vectors for gene therapy

Over the past years, a wide range of vector systems of viral and nonviral origin have been developed. While methods such as the direct injection of naked plasmid DNA, gene transfer through a gene gun, or as liposome vesicle showed low transfection efficiency, experiments using viral vectors looked more promising.

Figure 3.3. Figure 3.3. Liposomes

Figure 3.3. Liposomes

Figure 3.4. Figure 3.4. Naked DNA

Figure 3.4. Naked DNA

Figure 3.5. Figure 3.5. Gene particle bombardment

Figure 3.5. Gene particle bombardment

Genomes of retroviruses, adenoviruses and adeno-associated viruses (AAV) make up the bulk of the most frequently used viral vectors. Other, less commonly used viral vectors are derived from the Herpes simplex virus I (HSV-1), the baculovirus, and others. Viruses have evolved and adopted many properties of cells in the process, which enables them to identify target cells efficiently and penetrate them. They migrate from the cytoplasm to the nucleus to express their genes in the host cell. This viral life cycle enables infectious virions to transfer genetic information with great success.

Retroviral Vectors

Retroviruses are a large versatile group of viruses with a genome consisting of single or double-stranded RNA. They have a diameter of about 100 nm and they are covered by an envelope. The envelope contains a viral glycoprotein which binds to cellular receptors, thus defining the specificity of the host and cell type that is infected. The envelope protein furthers fusion with a cellular surface membrane or with endosomal compartments inside the cell. Depending on the arrangement of their genome, retroviruses are divided into two categories, the simple and the complex retroviruses. All retroviruses contain three essential genes, gag, pol, and env. Gag codes for structural proteins that constitute the matrix, the capsid, and the nucleoprotein complex. Pol codes for reverse transcriptase and integrase, while env codes for the proteins of the envelope. There is also a psi packaging signal and two LTRs (long terminal repeats) with regulatory functions within the virus. The prototype for a simple retrovirus carrying only a small set of information is the Moloney murine leukemia virus (MoMLV). Complex retroviruses such as lentiviruses (e.g., the human immunodeficiency virus, HIV) contain additional regulatory and accessory genes. Initially, vectors for gene therapy were developed from simple retroviruses, very often MoMLV. In order to develop retroviral vectors, knowledge of the viral life cycle became fundamental. After the infection of the host cell, the viral RNA is reversely transcribed into linear double-stranded DNA by reverse transcriptase. This process takes place in the cytoplasm, and the viral DNA is then introduced into the nucleus followed by a stable insertion into the host genome.

Figure 3.6. Figure 3.6. Retrovirus

Figure 3.6. Retrovirus

Figure 3.7. Figure 3.7. Life cycle of retroviruses

Figure 3.7. Life cycle of retroviruses

Figure 3.8. Figure 3.8. Moloney Murine Leukemia Virus Based Retroviral Vector I.

Figure 3.8. Moloney Murine Leukemia Virus Based Retroviral Vector I.

The mechanism by which retroviruses are introduced into the nucleus of the host cell differs between simple and complex retroviruses. Whereas simple retroviruses can only enter the nucleus where the nuclear membrane is being dissolved during the mitotic process, lentiviruses have a pre-integration complex that relies on an active cellular transport mechanism through the nuclear pore without destroying the nuclear membrane. Unlike MoMLV, lentiviruses are therefore able to transduce stationary host cells. Once the virus has entered the nucleus, the viral enzyme integrase initiates the integration of the viral DNA into the host genome. The integrated viral DNA is called a provirus. It limits expression of cellular genes and uses the host cell for viral gene expression. The transcriptional activity of the host is controlled by cis-acting proviral LTR regions. Complex retroviruses have additional trans-acting factors that activate RNA transcription (e.g., HIV-1 tat). After the translation of the viral genes, the resulting protein products and the viral RNA form viral particles that are released from the cell via the cell membrane by budding.

Most retroviral vectors currently used in gene therapy studies are based on MoMLV, one of the first gene vehicles used in human gene therapy experiments. In order to produce viruses with a deficient replication mechanism that only replicate in the packaging cell and not in the host cell, the viral genes have been removed and replaced by a therapeutic gene. Gag, pol, and env are expressed in trans in the packaging cell. When the modified viral genome containing the therapeutic gene is transfected into the packaging cell, all required components are brought together to form a recombinant virus. This virus can transfect target cells, but is unable to form infectious particles because genes code for viral proteins are missing from its genome. This is a safety device often used in viral vectors. The viral genes responsible for the replication of the virus are separated from the rest of the genome, thus reducing the risk of a recombination of infectious particles.

Figure 3.9. Figure 3.9. Moloney Murine Leukemia Virus Based Retroviral Vector II.

Figure 3.9. Moloney Murine Leukemia Virus Based Retroviral Vector II.

Lentiviruses are a subfamily of retroviruses with all the advantages of retroviral constructs plus the ability to transduce also postmitotic cells and tissue, including neurons, retinal, muscle, and hematopoietic cells. Recently developed lentiviral vectors are largely based on the HIV genome. In order to avoid a recombination of infectious HIV particles, as many endogenous HIV proteins as possible have been deleted without reducing the transduction and expression rate. Furthermore, more recently developed vectors carry regulatory elements that have been added later. The cPPT (central polypurine tract) sequence facilitates the synthesis of the second strand and the transport of the pre-integration complex into the nucleus, while the WPRF (woodchuck hepatitis virus posttranscriptional regulatory element) sequence enhances the expression of the transgene via a higher efficiency of the transduction and translation processes. An additional mutation of the 3’-LTRs results in self-inactivation (SIN), thus reducing the risk of a recombination of infectious HIV particles. In order to improve safety further, efficient vectors have been developed from lentiviruses that are not pathogenic to humans, but also have the ability to transduce stationary cells. The basic structure comes from, e.g., the monkey-specific simian immunodeficiency virus (SIV), the cat-specific feline immunodeficiency virus (FIV), or the horse-specific equine infectious anemia virus (EIAV). So far, most lentiviral vectors have been produced through transient transfection of packaging and vector plasmids. The cells used are the easily transfectable 293T cells, which yield titers between 1 x 109 and 1 x 1010 infectious units per mL. The virus particles are further concentrated through ultracentrifugation. However, it is not easy to standardize virus production in transient infections. It would be an advantage to be able to develop stable production cells, especially in view of the use of HIV-based vectors in clinical trials, but the toxicity of the VSV-G envelope protein and of other lentiviral proteins such as Gag, and Tat is a major obstacle in the production of lentiviral packaging cell lines. It would be useful if these toxic proteins could be expressed in tetracyline-regulated systems, a strategy currently under investigation. The potential risk for the application of lentiviral vectors lies in the possibility of insertion mutagenesis and a strong tendency in retroviruses to recombine with infectious foreign retroviruses either within the transfected or the target cells. It is also possible that new viruses emerge through recombination with endogenous sequences. Thus, new infectious viruses with hitherto unknown properties could be spread by the use of retroviral vectors. These could not only affect other organs, but also the germ cells.

Figure 3.10. Figure 3.10. Retroviral gene therapy

Figure 3.10. Retroviral gene therapy

Figure 3.11. Figure 3.11. Lentiviral vector

Figure 3.11. Lentiviral vector

Adenoviral Vectors

Until recently, adenoviral viruses were very popular because they can be easily produced on an industrial scale, the virus titers are high and they can transfect stationary as well as dividing cells. The linear double-stranded DNA of adenoviruses codes for 11 proteins. The genome is packed into an icosahedral protein capsule, which is not surrounded by an envelope, but contains fiber envelope proteins. The fiber proteins combine with the surface receptors of the host cell to form a high-affinity complex. The endosomes are lysed by the adenoviral enzymes, but the genome is not inserted into the cell DNA and remains episomal. This results in a serial dilution of the adenoviral genome over several cell divisions. Unlike retroviruses, adenoviruses cannot be passed on via the germ line. Their high expression rate on a short-term basis makes them suitable for tumor treatment. Due to their wide host tropism adenoviral viruses are not restricted to one compartment but spread into surrounding tissue. This leads to toxic side effects particularly on the liver. Furthermore most patients have already been exposed to adenoviruses during their lifetime and thus developed antibodies, which makes therapeutically relevant target tissues such as the epithelium of the respiratory tract as well as various tumors refractory to an adenoviral infection. This could reduce the efficacy of adenoviral gene therapy. What is more, conventional adenoviral vectors could elicit a strong immune reaction in the host mainly caused by the adenoviral E2 protein. While such an inflammatory reaction might well have an antitumor effect, there is also a high safety risk as the death of a patient has demonstrated. The replication defect in first generation adenoviral vectors was the result of a deletion of the E1A and E1B genes. In some of these vectors, the E3 gene was also deleted in order to improve their uptake capacity. However, they retain the other early and late viral genes that are expressed in small quantities after infection. In second generation adenoviral viruses where the E2 and E4 regions have also been deleted, and only the late genes are retained. Viral gene products induce an immune response against the transduced cells, resulting in a reduced expression of the transgene. New strategies aim to completely avoid the immune response and to achieve a higher uptake capacity for foreign DNA in adenoviral viruses. This led to the development of adenoviral vectors in which all viral reading frames have been deleted. These are known as gutless vectors and contain only those viral DNA sequences that are active in cis and are essential for the replication and packaging of viral DNA, such as inverse terminal repeats (ITRs), which contain the polymerase binding sequence for the start of DNA replication and the DNA packaging signal pis. The original adenoviral gene region between the two ITRs has been replaced by foreign non-coding DNA. In recombinant vectors derived from gutless vectors, this space is partially taken by the transgene. Gutless vectors can only be produced with the assistance of a helper virus, which provides the proteins required for viral replication and packaging.

Figure 3.12. Figure 3.12. Adenoviruses

Figure 3.12. Adenoviruses

Figure 3.13. Figure 3.13. Evolution and adenoviral vectors

Figure 3.13. Evolution and adenoviral vectors

Figure 3.14. Figure 3.14. Adenoviral gene therapy

Figure 3.14. Adenoviral gene therapy

Adeno-Associated Virus (AAV)

The adeno-associated virus (AAV), member of the parvovirus family, is a new promising candidate for the transfer of genes. AAV has an icosahedral structure and contains a single-stranded DNA genome of only 4.7 kb. It can only be replicated with the assistance of helper viruses such as adenoviruses or herpes viruses. Although a large proportion of the population is AAV-seropositive, so far no pathogenicity has been observed. In contrast to adenoviruses, AAV are only weakly immunogenic. They can infect dividing as well as stationary cells and integrate into the host genome, which is advantageous far long term expression. Wild type AAV contains no more than two genes, rep for replication and cap for encapsulation. The coding sequences are flanked by ITRs, which are needed for packaging DNA into capsids. In AAV vectors, the genes rep and cap have been replaced by a therapeutic gene. In order to produce a recombinant virus, the AAV genes and adenoviral helper genes are expressed in a packaging cell in trans. The major advantage of AAV-derived vectors is the ability to stably integrate into the target cell genome at a defined location in the chromosome. Location-specific insertion is mediated by a usually inactive 100 bp long region in the REP protein. However, since AAV vectors no longer contain the rep gene, targeted integration could only be detected using wild type AAV. Furthermore, since AAV is widespread among the human population, the question arises if the AAV-specific insertion location has been occupied by other genetic material. This would have to be removed first before an AAV vector could be used efficiently. It is also unclear what would happen if the insertion location of an AAV-derived vector were not available - whether sequence-independent insertion or even chromosomal relocation would take place. Another interesting property in AAV vectors derives from their specific chromosomal insertion: the capacity of homologous recombination. It was also possible to correct point mutations and deletions using an AAV vector, albeit at a very low frequency. This approach might also hold promising therapeutic possibilities.

Figure 3.15. Figure 3.15. Adeno-associated viruses

Figure 3.15. Adeno-associated viruses

At this stage, the production of AAV vectors is still a major problem, being very difficult and time-consuming. The rep gene and some of the adenoviral helper genes are cytotoxic to packaging cells, and there are no cell lines available for the large-scale production of pure recombinant viruses. Despite many limitations, AAV vectors are quite useful gene transfer systems, since they achieve excellent expression in muscle, brain, hematopoietic precursor cells, neurons, photoreceptor cells, and hepatocytes.