Lentiviral Vectors in Gene Therapy

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A Viral Biorealm page on the family Lentiviral Vectors in Gene Therapy

Lentiviruses are some of the most infamous–and life threatening–vertebrate viruses. Viruses in this genus include human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). Their exogenous characteristics and successful gene integration properties make them incredibly ideal for use in gene therapy.
Gene therapy requires delivery vehicles to transfer a gene into a target cell's chromosome and the success of treatment depends largely on the vector available to deliver therapeutic genes. Lentiviral vectors are highly successful in crossing the nuclear membrane and permanently changing the target cell, thus potentially increasing the efficacy and longevity of therapeutic treatment. Hemophilia and other blood-related diseases, AIDS, and various cancers are current targets of lentiviral vectors and they can be used in conjunction with other antiviral and anticancer drugs. The study of viral vectors in gene therapy is a fairly new field, with viral vectors being introduced in 1968 [23] but the progress that has been made is astounding. Lentiviral vectors can be used to specifically target cells and treat cancer tumors, HIV-infected T-cells, inherited diseases; they can also induce the transduced cells to amplify their protein production and to make transgenic mice for modeling. A variety of non-viral and viral vectors are in use to deliver therapies but lentiviral vectors are unanimously the most successful.


Contents

Gene Therapy

Figure 1. A detailed example of ex vivo cell targeting [1].

Genetic disorders result from mutated or missing genes. The goal of gene therapy is to repair the flawed DNA by completely repairing it-in most cases the expectation is a total replacement of the gene. Reparation at the source of the disorder hopefully impacts subsequent cells and tissues down the line instead of simply treating the symptoms of the disorder. In order to repair the disorder at its source, there must be an efficient way to gain entry into the cells and permanently incorporate the new gene into the target cell's genome. Various methods exist for this delivery and success depends on the nature of the disorder. What genes are involved, how many genes are involved, how the patient's immune system will react, and the physical effects of the mutated or missing gene has on a person must all be taken into account when considering gene therapy as a treatment option. If a disorder can be rectified by replacing the mutated gene, and the target cells are fairly accessible, then a suitable method of gene delivery will be devised.

"Vectors" refer to the method of gene insertion. Non-viral vectors are plasmids cultivated in bacteria and naked plasmids that are inserted directly into the cell. Non-viral vectors are not cell specific but are generally less effective in their integration into the host cell [15]. Viral vectors refer to modified viruses used to deliver DNA into target cells. Viral vectors are usually more specific to certain cell types but are limited in the size of the genome they can fit inside their capsid. Viral vectors may cause an immune response in the patient, or if the patient is inherently immune to the specific type of virus, then the success of DNA integration is diminished.

There are also various ways in which target cells can be isolated for integration. Ex vivo requires a removal of target cells from the patient, growing them in a culture, and introducing the vector into the culture. After the target cells have integrated the DNA, they are replaced into the patient. In vivo treatment simply requires the injection of the vector that is specific to target cells into the patient. Since viral vectors are commonly regarded as the most effective delivery systems, many methods have been researched and utilize various types of viruses: retroviruses, adenoviruses, adeno-associated viruses, and the herpes simplex virus are common. Viruses can also be modified to express fewer capsid proteins and to express certain glycoproteins that are more specific to target cells.

Choosing the type of virus vector depends on the nature of the genetic reparation needed. For example, retrovirusesonly infect dividing cells but subsequent cell lineages will contain the inserted DNA. DNA carried by adenoviruses and the herpes simplex virus will not incorporate into the host cell's DNA and may be discarded or degraded by the host cell after a period of time. For this reason, focus has shifted into retroviral vectors, specifically lentiviruses.

About Lentiviruses

Retroviruses are characterized by their use of the host cell's reverse transcriptase to create DNA from viral RNA. The DNA is then incorporated into the host's DNA genome. Viruses in the genus Lentivirus have slow incubation periods and are categorized into five different serotypes based on their host: primates, sheep/goats, horses, cats, and cattle. RELIK was found to be the first endogenous lentivirus, infecting rabbits, dating millions of years [7]. Present lentiviruses are exogenous, incorporating their DNA into a host's upon infection, and they can infect non-dividing cells, unlike most other retroviruses.

After a long incubation period and subsequent cell divisions and virus gene proliferation, lentiviruses cause prolonged illness, such as AIDS in humans. Much is known about the method of infection of lentiviruses. Glycoproteins (gp) on the envelope of the virus recognize antibody receptors on host cells and attach to them, prompting membrane fusion of the two. Viral RNA is released from the capsid in the cytoplasm of the cell and host cell reverse transcriptase beings to synthesize complementary DNA. The double-stranded DNA is taken into the nucleus and integrated into the host's genome. It can lay dormant until necessary transcription factors are present to begin transcription of the viral DNA. As long as the viral DNA lays dormant within the host's genome, the host will duplicate it during cell division and all lineages thereafter will contain the viral DNA. When the viral DNA undergoes transcription, it will leave the nucleus and the cell's mechanisms will translate the mRNA into viral proteins, eventually assembling new virions that erupt from the cell, killing it.

Over time, viral DNA that has incorporated itself into the host's germline becomes endogenous to the host; human DNA is famously made up of at least 8% of endogenous retroviral DNA (that seem to have no known function). Retroviruses are also notorious for their high mutation rates during reverse transcription and this may cause complications when attempting to directly interfere with drug therapies targeted at specific viruses.

Figure 2. The retrovirus infection cycle. [2].


Lentiviral Vectors

Successful gene therapy requires permanent expression of the gene of interest in the target cell. The efficiency of the mode of gene delivery obviously correlates to the success of DNA integration into the host. Lentiviruses have been of interest for many years because of the notoriously successful infection rate of wild-type viruses. Lentiviruses can infect both dividing cells and non-dividing cells, a crucial criterion for many treatments that deal with stationary cell lines [10]. The long terminal repeat (LTR) region of HIV-1 is a target area for modification; the sequence is a mediator for virus DNA integration into host cells. Env proteins can be modified with ligands and antibodies to target a more specific cell type. In vivo cell targeting is certainly the most complicated of the delivery methods but can produce successful noninvasive results more quickly if honed.

One method of vector transduction involves specific antibodies on the envelope of the virus recognizing target cell surface molecules (such as αCD20 on the viral envelope recognizing CD20 on the target cell; CD20 is an antibody on a B-cell), and a separate, modified, fusion glycoprotein attaches to the host cell. Figure 3 shows an early, simple lentivirus vector and it’s cell-entry mechanism [21]. The virus is taken into the cell via endocytoses. The virus is within an endosome and fuses to its membrane, prompting the viral and endosome membranes to open and the capsid is released into the cell. From there, the virus 'infects' the host cell as per usual and the therapeutic gene is incorporated into the target cell.

Vectors are packaged into viral structures with post-transcriptionally active elements designed to regulate and enhance transgene expression [11]. These helper packaging constructs contain important promoters, glycoproteins that broaden the range of host recognition (changing the glycoproteins on a vector means creating a pseudotype), and non-human-specific virus sequences to prevent recombination and vector mobilization. Woodchuck hepatitis virus is a very commonly used element, as well as other plant elements, rhabdoviruses, retroviruses, and arenaviruses. The one created for the HIV lentivirus VRX496, called VIRPAC (as seen in the bottom half of Figure 5) contains a rhabdovirus VSV-G glycoprotein.

Figure 3. A supplementary figure from Yang, et al, 2003, showing the entry method of their lentiviral vector. [3].


Figure 4. The structure of a typical lentiviral vector, looking much like a wild-type virus particle. [4].


Current Lentiviral Treatments

HIV
In one study, patients who were resistant to several combinations of drug therapies were given one dose of an ex vivo treatment of a lentiviral vector named VRX496 containing an anti-sense gene against the HIV envelope protein and intact LTRs [9] and monitored for several months. When the vector gene is integrated into the patient’s T-cells, RNA created from the antisense gene will block mRNA created by infectious HIV, blocking the mRNA and preventing the cell to continue making envelope proteins for the virus. Viral load, CD4 count, and other immunological factors were tested. Four out of the five patients showed overall improvement in the reduction of their viral load, had increased CD4+ T-cell counts, and increased immune response to HIV antigens [9]. VRX496 is a conditionally replicating vector, meaning that transcription is amplified in the presence of wilt-type HIV. As a result, conditionally replicating vectors are less inclined to transfect non-target cells and are “self-propagating” [Connolly]. These patients were treated in 2006 and are entered in Phase 2 of clinical trials, pending results for a second infusion of VRX496 T-cells [20].

Patients who are undergoing successful HAART (highly active antiretroviral therapy) were also given a dose of VRX496 and monitored for CD4+ counts and viral load. Phase 2 of the study requires the patients to cease HAART and will be monitored for five years [19]. Final data will be available in 2020.

Other RNA-interfering vectors being used in HIV patients to suppress wild-type HIV replication are being tweaked to target either the HIV genome or the mRNAs which the host cell creates [3].

Figure 5. A figure (1A) from Levine, et al, 2006, showing the constructed VRX496 vector [5].

X-Linked Disease
Adrenoleukodystrophy (ALD) affects males in that they cannot metabolize very long chained fatty acids. The accumulation of these fatty acids can lead to nervous system deterioration (as severe as entering into an untreatable coma), paralysis, and progressively weakening muscles [1]. The ABCD1 gene encodes the ALD proteins that are transporters in the membrane of peroxisomes which normally metabolize the very long chain fatty acids and a mutation of or deletions in ABCD1 disrupts this process. Cell transplantation is the current treatment, but it must be administered at an early age. Lentiviral vectors aim to transduce hematopoietic stem cells that results in long-term expression of the corrected ALD gene in resulting hematopoietic cell lines [5]. Two children who had no matching donors for cord blood or human leukocyte antigen were given ex vivo doses of an HIV-derived lentivirus containing wild-type ABCD1. Many transduced CD4+ T-cells expressed ALD proteins and could successfully metabolize very long chained fatty acids. After time, expression decreased but stabilized in blood cell lines [5].

Cancers
The selectivity of vectors makes them ideal for using them to target tumor cells. Tumors may be made up of many types of cells, however, and the efficacy of using only one selective vector is low. There is also some concern about the unknown effects of chemotherapy use in conjunction with vectors [6]. Increasing the amount of vectors in a dose decreases their selectivity and the risk of non-tumor vector infection is raised. Vector replication is limited in mouse models and their spread is unsuccessful [6], and repeated exposure to vectors could cause averse immune reactions and impediments. Vectors can also contain pro-drug activating enzymes to increase anti-tumor drug efficacy in patients. Creating replication-deficient vectors is easily done by removing the viral gag, pol, and env genes, among others, and replacing them with a cassette that contains the desired genes; the removal of these genes also suppresses host immune response, but this creates an unstable vector that cannot transduce to other tumor cells [4]. Targeting specific tumor cells, as mentioned, is tricky because of the potentially heterogeneous cell makeup of the tumor. Several different tissue-specific vectors would have to be dosed with unknown potential side effects or reduced efficacy. Vectors have been successful in treating liver tumors caused by hepatitis B and C virus infections; hepatocellular carcinoma cells were transduced in vitro with a vector expressing herpes simplex virus thymidine kinase that induced apoptosis [18]. Other cancers have also been observed to regress [22].

Other
Lentiviral vectors are useful in transducing retinal cells in vivo [12], repopulating organs using blast cells ex vivo and transplanting them into the body [14], nervous system transductions [8], and many other curative and preventative treatments.

Potential Side Effects/Safety

Using foreign vectors in a patient always runs the risk of a severe adverse immune reaction. The "randomness" of the insertion of the lentiviral vector's DNA can potentially disrupt existing normal genes. Insertion of vector genes near patient oncogenes can result in the patient developing leukemia. Integration site analysis of vector genes shows that insertion sites of vectors are random but tend to integrate in gene-rich areas of chromosomes where those genes are highly expressed [9]. Integration analysis is done by cleaving sample DNA at a specific sequence with restriction enzymes. Synthesized ‘adaptor’ DNA is added to the end of the cleaved DNA and amplified with PCR primers complementary to the adaptor DNA end and primers complimentary to the vector DNA terminus end. The DNA bound to the vector DNA primer can be sequenced and identified [2]. Figure 6 shows the vector integration analysis results for VRX496 and that it is non-random, insterting itself into the patient's genome near other highly-expressed genes.
Virus proteins on its capsid can be removed and replaced by less-specific or less-selective protein. Once in the cell, "vector mobilization" may occur and infect other tissues besides the target. This increased tropism of the vector depends largely on the pseudotype of the vector and those using rhabdovirus-derived elements may have a larger target range than vectors with respirovirus elements, which target hepatocytes. An element that has a broader target range is more worrisome, though there are methods to track the vector in patient samples and determine if the vector has become replication competent. The vector cassette usually contains a marker sequence that can be assayed for; for example, VRX496 contains a Gtag segment (Figure 5) that is derived from GFP and its levels can be assayed. VRX496 was shown not to be replication competent and the Gtag also showed that transduced T-cells lived for close to 400 days after engraftment [9].

Cancerous versions of cells are specifically targeted but their vectors must be carefully developed. If a vector is targeting hepatocellular carcinoma cells then they must also not be able to easily transduce normal hepatocyte cells. The type of promoter packaged with the vector is important in determining the mobility between closely related cells. It may also be possible to transduce both cancerous cells and normal cells with the same vector, but while the cancerous cells are being destroyed, the normal cells have a target marker silenced so they do not become transduced [18].

Figure 6. A figure (3A) from Levine, et al, 2006, detailing the site integration analysis for VRPX496. Blue sticks mark vector entry sites and red marks denote gene density. The more gene-rich an area is, the more dense the red coloring. [6].

Future Directions of Lentiviral Vectors

Because of the unique ability that allows lentiviral vectors to transduce both dividing and non-dividing cells, many treatment options and combinations are possible. Lentiviral vectors are also more long-term than many of the other viral vectors [12]. In vivo injections of vectors are slightly more inefficient treatments due to the large amounts of the vector needed and the exposure of other organs to the vector. Non-systemic ex vivo treatments have been highly successful, barring the sometimes limited amount of cells that can be sampled, and long-term and research may lean towards perfecting this method.

Transducing stem cell lines with certain genes may “recruit” cells derived from cell lines. Ailments affecting the central nervous system including the brain can be treated with in vivo transductions [8] and bone-marrow derived cells may be recruited to become macrophages. More specific promoters within vectors can tailor them to be highly selective to only certain types of cells with very low risk for mobility or increased tropism. Many diseases also must be treated in their early-stages and if there are inadequate treatments or health providers, a patient may simply lose the chance at recovery because their illness was not caught early enough. The effectiveness of lentiviral vectors may make it so that diseases can be treated at later stages with increased efficacy. Parkinson disease, Huntington disease, and other inherited diseases are of interest to be treated with lentiviral vectors.

As with any market, drug production is a balance between safeties, cost, and time. Successful trials on rats, mice, and in vitro cells is only the first step in a long line of drug production. Clinical trials take years to complete and must be performed precisely. Biotechnology companies are investing in vector production and are realizing the potential of creating viral vectors that maximize cell protein production, to creating more specific vaccines, to creating specific lentiviral vectors that express the genes to generate induced pluripotent stem cells [16] are being made. Untapped potential lies in these small particles and because of their versatility; they have a broad range of applications.

Because of their reliable gene integration, ease of use, and longevity, lentiviral vectors will undoubtedly continue to be an area of gene therapy research that thrives. There are many, many, applicable uses for lentiviruses (such as being used to insert markers into cells and geneomes of interest for tracking use) and many iterations of the vector. Using it in combination with other treatments and drugs is still yet to be totally explored. It is interesting and important to consider the exciting applications and perhaps lentiviral vectors will become so important, and ubiquitous, that they will be cheaper than they are today-vectors for research purposes can cost up to $625.

References

[1] A.D.A.M. Medical Encyclopedia [Internet]. Atlanta (GA): A.D.A.M., Inc.; c1997-2011. Adrenoleukodystrophy [updated 2011 Dec; cited 2012 Dec] Available from: <http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0002165/>
[2] Brady, T., S.L. Roth, N. Malani, et al. 2011. "A method to sequence and quantify DNA integration for monitoring outcome in gene therapy." Nucleic Acids Research 1-8.
[3] ter Brake, O., J.T. Westerink, B. Berkhout. 2010. "Lentiviral vector engineering for anti-HIV RNAi gene therapy." Lentivirus Gene Engineering Protocols, Methods in Molecular Biology 614:201-213.
[4] Breckpot, K., J.L. Aerts, K. Thielemans. 2007. "Lentiviral vectors for cancer immunotherapy: transforming infectious particles into therapeutics." Gene Therapy 14: 847-862.
[5] Cartier, N., S. Hacein-Bey-Abina, C.C. Bartholomae, et al. 2009. "Hematopoietic stem cell gene therapy with a lentiviral vector in X-Linked Adrenoleukodystrophy." Science 326: 818-823.
[6] Connolly, J.B. 2003. "Conditionally replicating viruses in cancer therapy." </i>Gene Therapy</i> 10: 712-715.
[7] Katzourakis, A., M. Tristem, O. G. Pybus, R. J. Gifford. 2007. "Discovery and analysis of the first endogenous lentivirus." Proceedings of the National Academy of Sciences of the United States of America. 104: 6261-6265.
[8] Lai Z., R.O. Brady. 2002. "Gene transfer into the central nervous system in vivo using a recombinant lentivirus vector." J Neurosci Res 67: 363-371.
[9] Levine, B.L., L.M. Humeau, J. Boyer. et al. 2006. "Gene transfer in humans using a conditionally replicating lentiviral vector." PNAS 103: 17372-17377.
[10] Lewis, P.F., M. Emerman. 1994. "Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus." J of Virology 68: 510-516.
[11] Mastroyiannopoulos NP, Feldman ML, Uney JB, Mahadevan MS, Phylactou LA. 2005. "Woodchuck post-transcriptional element induces nuclear export of myotonic dystrophy 3' untranslated region transcripts." EMBO Rep 6: 458-463.
[12] Hiroyuki M., M. Takahashi, F.H. Gage, I.M. Verma. 1997. "Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector." PNAS 94: 10319-10323.
[13] Naldini, L. 1098. "Lentiviruses as gene transfer agents for delivery to non-dividing cells." Current Opinion in Biotechnology. 9: 457-463.
[14] Oertel, M., R. Rosencrantz, Y.Q. Chen, et al. 2003. "Repopulation of Rat Liver by Fetal Hepatoblasts and Adult Hepatocytes Transduced Ex Vivo With Lentiviral Vectors." Hepatology 5: 994-1005.
[15] Somia N. V. , Zoppe M. , Verma I. M. 1995. "Generation of targeted retroviral vectors by using single-chain variable fragment: an approach to in vivo gene delivery." Proc. Natl. Acad. Sci. USA 92:7570–7574.
[16] "Stem Cell Therapies Overview." Lentigen Corporation. 2012. <http://www.lentigen.com/products/stemcell-overview>
[17] "Tools of the Trade." Learn Genetics. Genetic Science Center, University of Utah, n.d. Web. Nov. 2012. <http://learn.genetics.utah.edu/content/tech/genetherapy/gttools/>.
[18] Uch, R., Gerolami R, Faivre J, Hardwigsen J, et al. 2003. "Hepatoma cell-specific ganciclovir-mediated toxicity of a lentivirally transduced HSV-TkEGFP fusion protein gene placed under the control of rat alpha-fetoprotein gene regulatory sequences." Cancer Gene Ther 10: 689–695.
[19] University of Pennsylvania, Pennsylvania. Evaluate the Tolerability and Therapeutic Effects of Repeated Doses of Autologous T-Cells with VRX496 in HIV. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2000- [cited 2012 Dec]. Available from: <http://www.clinicaltrials.gov/ct2/show/study/NCT00295477?term=VRX496&rank=2>
[20] VIRxSYS Corporation. A Rollover Study for Subjects Who Completed Participation in the VRX496-USA-05-002 Trial. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2000- [cited 2012 Dec]. Available from: <http://www.clinicaltrials.gov/ct2/show/NCT00622232?term=VRX496&rank=1>
[21] Yang, L., L. Bailey, D. Baltimore, P. Wang. 2006. "Targeting lentiviral vectors to specific cell types in vivo." Proc. Natl. Acad. Sci. USA 103:11479-11484.
[22] Zheng, J.Y., D. Chen, J. Chan, et al. 2003. "Regression of prostate cancer xenografts by a lentiviral vector specifically expressing diptheria toxin A." Cancer Gene Therapy 10: 764-770.
[23] Walther, W., U. Stein. 200. "Viral Vectors for Gene Transfer." Drugs 60: 249-271.

Page authored by Irene McIntosh for BIOL 375 Virology, December 2012

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