Zinc finger nuclease treatment of HIV

Last updated

Since antiretroviral therapy requires a lifelong treatment regimen, research to find more permanent cures for HIV infection is currently underway. [1] It is possible to synthesize zinc finger nucleotides with zinc finger components that selectively (almost selectively) bind to specific portions of DNA. Conceptually, targeting and editing could focus on host cellular co-receptors for HIV or on proviral HIV DNA.

Contents

Host cellular co-receptors for HIV

It has also been observed that 20% of the Caucasian population possess a mutation, called CCR5-Δ32 (frequency of 0.0808 for homozygous allele), that prevents the CCR5 chemokine receptor protein, which is the main means of viral access into the cell, from being expressed on the surface of their CD4 + T-cells. [2] [3] [4] [5] [6] Individuals who are homozygous for this mutation are immune to HIV strains that use the CCR5 receptor to access the cell, while those that are heterozygous for this mutation have been found to reduce plasma viral load and delay progression of AIDS. [7] [8] By combining these facts, researchers have proposed a novel method of treatment for HIV. This method attempts to treat the infection by disrupting the CCR5 gene, such as introducing the CCR5-Δ32 mutation using a recombinant adenoviral vector or forcing DNA repair by nonhomologous end joining, which is prone to error and results in a non-functional gene. As a consequence, resulting in the expression of nonfunctional CCR5 co-receptors on CD4+ T cells, providing immunity against infection. [9] [7] [10] [11]

The zinc finger nucleases that have been synthesized for this treatment are manufactured by combining FokI Type II restriction endonucleases with engineered zinc fingers. [9] [12] The number of zinc fingers attached to the endonuclease controls the specificity of the ZFN since they are engineered to preferentially bind to specific base sequences in DNA. Each ZFN is made up of multiple zinc fingers and one nuclease enzyme. [9]

Proviral HIV DNA

A recent and unique application of ZFN-technology to treat HIV has emerged whose focus is to target not the host genome, but rather proviral HIV DNA, for mutagenesis. [13] The authors of this work have drawn their inspiration from the innate defense mechanism against bacteria-infecting-viruses called bacteriophages, present amongst those bacteria endowed with restriction modification (R-M) systems. These bacteria secrete a restriction enzyme (REase) that recognizes and repetitively cleaves around palindromic sequences within the xenogenic DNAs of the bacteriophages or simply phages, until the same is disabled. Further support for this approach resides in the fact that, the human genome comprises in large part remnants of retroviral genomes that have been inactivated by several mechanisms, some of whose action resembles that of ZFN. It should not be surprising, therefore, that the initial work leading to the application of ZFN technology in this manner revolved around and involved the isolation and testing of HIV/SIV targeting bacteria-derived REases, whose non-specificity (due to their short recognition sequences) unfortunately, rendered them toxic to the host genome. The latter-potential host-genome toxicity posed by the raw bacteria-derived REases limited their application to ex-vivo modalities for HIV prevention, namely synthetic or live microbicides. Subsequently, however, the unique specificity offered by ZFNs was quickly recognized and harnessed, paving way for a novel strategy for attacking HIV in-vivo (through target mutagenesis of proviral HIV DNA) that is similar to the manner by which bacteria equipped with R-M systems do, to disable the foreign DNAs of in-coming phage-genomes. Because latent proviral HIV DNA resident in resting memory CD4 cells forms the major barrier to the eradication of HIV by highly active antiviral therapy (HAART), it is speculated that this approach may offer a 'functional cure" for HIV. Both ex-vivo (manipulation of stem or autologous T cell precursors) and in-vivo delivery platforms are being explored. It is also hoped that, when applied to non-HIV infected persons, this strategy could offer a genomic vaccine against HIV and other viruses. Similar work is ongoing for high-risk HPVs (with the intent of reversing cervical neoplasia) [14] as well as with HSV-2 (with the goal of achieving a complete cure for genital herpes) [15] [16] [17] [18] [19] [20] [21] [22] [23]

Zinc finger binding

The FokI catalytic domain must dimerize to cleave the DNA at the targeted site, and requires there to be two adjacent zinc finger nucleases (see picture), which independently bind to a specific codon at the correct orientation and spacing. As a result, the two binding events from the two zinc finger nuclease enables specific DNA targeting. [24] Specificity of genome editing is important for the zinc finger nuclease to be a successful application. The consequence of off-targeting cleavage can lead to a decrease in efficiency of the on-target modification in addition to other unwanted changes. [24]

The exact constitution of the ZFNs that are to be used to treat HIV is still unknown. The binding of ZFNs for the alteration of the Zif268 genelink, however, has been well-studied and is outlined below to illustrate the mechanism by which the zinc finger domain of ZFNs bind to DNA. [25] [26]

The amino terminus of the alpha helix portion of zinc fingers targets the major grooves of the DNA helix and binds near the CCR5 gene positioning FokI in a suitable location for DNA cleavage. [9] [25] [26]

Zinc fingers are repeated structural protein motifs with DNA recognition function that fit in the major grooves of DNA. [25] Three zinc fingers are positioned in a semi-circular or C-shaped arrangement. [26] Each zinc finger is made up of anti-parallel beta sheets and an alpha helix, held together by a zinc ion and hydrophobic residues. [25] [26]

The zinc atom is constrained in a tetrahedral conformation through the coordination of Cys3, Cys6, His19, and His23 and Zinc – Sulfur bond distance of 2.30 +/- 0.05 Angstroms and Zinc – Nitrogen bond distances of 2.0 +/- 0.05 Angstroms. [26] [27] [28]

Each zinc finger has an arginine (arg) amino acid protruding from the alpha helix, which forms a hydrogen bond with Nitrogen 7 and Oxygen 6 of the guanine (gua) that is located at the 3’ end of the binding site. [25] [26] [28] The arg-gua bond is stabilized by aspartic acid from a 2nd residue, which positions the long chain of arginine through a hydrogen bond salt bridge interaction. [25] [29]

In residue 3 of the 2nd (i.e., middle) zinc finger, histidine49 forms a hydrogen bond with a co-planar guanine in base pair 6. The stacking of Histidine against Thymine in base pair 5 limits the conformational ability of Histidine49 leading to increased specificity for the histidine-guanine hydrogen bond. [25] [26]

At the 6th residue, fingers 1 and 3 have arginine donating a pair of charged hydrogen bonds to Nitrogen 7 and Oxygen 6 of guanine at the 5’ end enhancing the site recognition sequence of zinc fingers. [25] [26]

Contacts with DNA backbone

The histidine coordinated to the zinc atom, which is also the seventh residue in the alpha helix of the zinc fingers, coordinates the Zinc ion through its Nε and hydrogen bonds with phosphodiester oxygen through Nδ on the primary DNA strand. [25] [26] [29]

In addition to histidine, a conserved arginine on the second beta strand of the zinc fingers makes contact with the phosphodiester oxygen on the DNA strand. [25] [26] [29]

Also serine 75 on the third finger hydrogen bonds to the phosphate between base pairs 7 and 8, as the only backbone contact with the secondary strand of DNA. [25] [26] [29]

Nuclease dimerization and cleavage

It has been discovered that FokI has no intrinsic specificity in its cleavage of DNA and that the zinc finger recognition domain confers selectivity to zinc finger nucleases. [9] [12]

Specificity is provided by dimerization, which decreases the probability of off-site cleavage. Each set of zinc fingers is specific to a nucleotide sequence on either side of the targeted gene 5-7 bp separation between nuclease components. [9]

The dimerization of two ZFNs is required to produce the necessary double-strand break within the CCR5 gene because the interaction between the FokI enzyme and DNA is weak. [11] This break is repaired by the natural repair mechanisms of the cell, specifically non-homologous end joining. [11]

Introducing the CCR5 mutation

Introducing genome alterations depends upon either of the two natural repair mechanisms of a cell: non-homologous end joining (NHEJ) and homology-directed repair (HDR). [11] Repair through NHEJ comes about by the ligation of the end of the broken strands and, upon the occurrence of an error, can produce small insertions and deletions. HDR, on the other hand, uses a homologous DNA strand to repair—and gene making use of this repair mechanism and providing the desired nucleotide sequence allows for gene insertion or modification. [11]

In the absence of a homologous nucleotide base sequence that can be used by a homologous recombination mechanism, the main DSB repair pathway in mammals is through non-homologous end joining (NHEJ). [30] NHEJ, although capable of restoring a damaged gene, is error-prone. [30] DSB are, therefore, introduced into the gene until an error in its repair occurs at which point ZFNs are no longer able to bind and dimerize and the mutation is complete. [30] To accelerate this process, exonucleases can be introduced to digest the ends of the strands generated at DSBs. [30]

Limitations

Increasing the number of zinc fingers increases the specificity by increasing the number of base pairs that the ZFN can bind to. [9] However too many zinc fingers can lead to off-target binding and thus offsite cleavage. [9] This is due to an increased likelihood of zinc fingers binding to parts of the genome outside of the gene of interest.

Current ZFN treatments focus on the CCR5 gene as no known side effects result from altering CCR5. [31] There are strains of HIV that are able to use CXCR4 to enter the host cell, bypassing CCR5 altogether. [31] The same gene editing technology has been applied to CXCR4 alone and in combination with CCR5 [32] [33]

Several issues exist with this experimental treatment. One issue lies in ensuring that the desired repair mechanism is the one that is used to repair the DSB following gene addition. [34] Another issue with the disruption of the CCR5 gene is that CXCR4-specific or dual-tropic strains are still able to access the cell. [34] This method can prevent the progression of HIV infection.

To employ the ZFNs in clinical settings the following criteria must be met:

i) High specificity of DNA-binding – Correlates with better performance and less toxicity of ZFNs. Engineered ZFNs take into account positional and context-dependent effects of zinc fingers to increase specificity. [35]

ii) Enable allosteric activation of FokI once bound to DNA in order for it to produce only the required DSB. [35]

iii) To deliver two different zinc finger nuclease subunits and donor DNA to the cell, the vectors that are used need to be improved to decrease the risk of mutagenesis. [35] These include adeno-associated virus vectors, integrase-deficient lentiviral vectors and adenovirus type 5 vectors. [35]

iv) Transient expression of ZFNs is preferred over permanent expression of these proteins to avoid ‘off-target’ effects. [35]

v) During gene targeting, genotoxicity associated with high expression of ZFNs might lead to cell apoptosis and thus needs to be thoroughly verified in vitro and in vivo transformation assays. [35]

Administration of treatment

The cells in which the mutations are induced ex vivo are filtered out from lymphocytes by apheresis to produce analogous lentiviral engineered CD4 + T-cells. [36] These are re-infused into the body as a single dose of 1 X 1010 gene modified analogous CD4 + T-cells. [36] A viral vector is used to deliver the ZFNs that induce the desired mutation into the cells. Conditions that promote this process are carefully monitored ensuring the production of CCR5 strain HIV-resistant T cells. [37]

The Berlin Patient

Timothy Ray Brown, who underwent a bone marrow transplant in 2007 to treat leukemia, had HIV simultaneously. [38] Soon after the operation the HIV dropped to undetectable levels. [38] This is a result of the bone marrow donor being homozygous for the CCR5-Δ32 mutation. [38] This new mutation conferred a resistance to HIV in the recipient, eventually leading to an almost complete disappearance of HIV particles in his body. [38] After nearly 2 years without antiretroviral drug therapy, HIV could still not be detected in any of his tissues. [38] [39] Though this method has been effective at reducing the level of infection, the risks associated with bone marrow transplants outweighs its potential value as a treatment for HIV. [3]

Related Research Articles

A restriction enzyme, restriction endonuclease, REase, ENase orrestrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone of the DNA double helix.

Gene knockouts are a widely used genetic engineering technique that involves the targeted removal or inactivation of a specific gene within an organism's genome. This can be done through a variety of methods, including homologous recombination, CRISPR-Cas9, and TALENs.

<span class="mw-page-title-main">Zinc finger</span> Small structural protein motif found mostly in transcriptional proteins

A zinc finger is a small protein structural motif that is characterized by the coordination of one or more zinc ions (Zn2+) which stabilizes the fold. It was originally coined to describe the finger-like appearance of a hypothesized structure from the African clawed frog (Xenopus laevis) transcription factor IIIA. However, it has been found to encompass a wide variety of differing protein structures in eukaryotic cells. Xenopus laevis TFIIIA was originally demonstrated to contain zinc and require the metal for function in 1983, the first such reported zinc requirement for a gene regulatory protein followed soon thereafter by the Krüppel factor in Drosophila. It often appears as a metal-binding domain in multi-domain proteins.

The restriction modification system is found in bacteria and other prokaryotic organisms, and provides a defense against foreign DNA, such as that borne by bacteriophages.

<span class="mw-page-title-main">Germline mutation</span> Inherited genetic variation

A germline mutation, or germinal mutation, is any detectable variation within germ cells. Mutations in these cells are the only mutations that can be passed on to offspring, when either a mutated sperm or oocyte come together to form a zygote. After this fertilization event occurs, germ cells divide rapidly to produce all of the cells in the body, causing this mutation to be present in every somatic and germline cell in the offspring; this is also known as a constitutional mutation. Germline mutation is distinct from somatic mutation.

<span class="mw-page-title-main">CCR5</span> Immune system protein

C-C chemokine receptor type 5, also known as CCR5 or CD195, is a protein on the surface of white blood cells that is involved in the immune system as it acts as a receptor for chemokines.

<span class="mw-page-title-main">Designer baby</span> Genetically modified human embryo

A designer baby is a baby whose genetic makeup has been selected or altered, often to exclude a particular gene or to remove genes associated with disease. This process usually involves analysing a wide range of human embryos to identify genes associated with particular diseases and characteristics, and selecting embryos that have the desired genetic makeup; a process known as preimplantation genetic diagnosis. Screening for single genes is commonly practiced, and polygenic screening is offered by a few companies. Other methods by which a baby's genetic information can be altered involve directly editing the genome before birth, which is not routinely performed and only one instance of this is known to have occurred as of 2019, where Chinese twins Lulu and Nana were edited as embryos, causing widespread criticism.

<span class="mw-page-title-main">CXCR4</span> Protein

C-X-C chemokine receptor type 4 (CXCR-4) also known as fusin or CD184 is a protein that in humans is encoded by the CXCR4 gene. The protein is a CXC chemokine receptor.

Entry inhibitors, also known as fusion inhibitors, are a class of antiviral drugs that prevent a virus from entering a cell, for example, by blocking a receptor. Entry inhibitors are used to treat conditions such as HIV and hepatitis D.

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. Alongside CRISPR/Cas9 and TALEN, ZFN is a prominent tool in the field of genome editing.

<span class="mw-page-title-main">Gene targeting</span> Genetic technique that uses homologous recombination to change an endogenous gene

Gene targeting is a biotechnological tool used to change the DNA sequence of an organism. It is based on the natural DNA-repair mechanism of Homology Directed Repair (HDR), including Homologous Recombination. Gene targeting can be used to make a range of sizes of DNA edits, from larger DNA edits such as inserting entire new genes into an organism, through to much smaller changes to the existing DNA such as a single base-pair change. Gene targeting relies on the presence of a repair template to introduce the user-defined edits to the DNA. The user will design the repair template to contain the desired edit, flanked by DNA sequence corresponding (homologous) to the region of DNA that the user wants to edit; hence the edit is targeted to a particular genomic region. In this way Gene Targeting is distinct from natural homology-directed repair, during which the ‘natural’ DNA repair template of the sister chromatid is used to repair broken DNA. The alteration of DNA sequence in an organism can be useful in both a research context – for example to understand the biological role of a gene – and in biotechnology, for example to alter the traits of an organism.

Zinc finger protein chimera are chimeric proteins composed of a DNA-binding zinc finger protein domain and another domain through which the protein exerts its effect. The effector domain may be a transcriptional activator (A) or repressor (R), a methylation domain (M) or a nuclease (N).

Recombinant adeno-associated virus (rAAV) based genome engineering is a genome editing platform centered on the use of recombinant AAV vectors that enables insertion, deletion or substitution of DNA sequences into the genomes of live mammalian cells. The technique builds on Mario Capecchi and Oliver Smithies' Nobel Prize–winning discovery that homologous recombination (HR), a natural hi-fidelity DNA repair mechanism, can be harnessed to perform precise genome alterations in mice. rAAV mediated genome-editing improves the efficiency of this technique to permit genome engineering in any pre-established and differentiated human cell line, which, in contrast to mouse ES cells, have low rates of HR.

<span class="mw-page-title-main">Transcription activator-like effector nuclease</span>

Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain. Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. The restriction enzymes can be introduced into cells, for use in gene editing or for genome editing in situ, a technique known as genome editing with engineered nucleases. Alongside zinc finger nucleases and CRISPR/Cas9, TALEN is a prominent tool in the field of genome editing.

<span class="mw-page-title-main">Genome editing</span> Type of genetic engineering

Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site-specific locations. The basic mechanism involved in genetic manipulations through programmable nucleases is the recognition of target genomic loci and binding of effector DNA-binding domain (DBD), double-strand breaks (DSBs) in target DNA by the restriction endonucleases, and the repair of DSBs through homology-directed recombination (HDR) or non-homologous end joining (NHEJ).

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

<span class="mw-page-title-main">HIV/AIDS research</span> Field of immunology research

HIV/AIDS research includes all medical research that attempts to prevent, treat, or cure HIV/AIDS, as well as fundamental research about the nature of HIV as an infectious agent and AIDS as the disease caused by HIV.

A small proportion of humans show partial or apparently complete innate resistance to HIV, the virus that causes AIDS. The main mechanism is a mutation of the gene encoding CCR5, which acts as a co-receptor for HIV. It is estimated that the proportion of people with some form of resistance to HIV is under 10%.

Off-target genome editing refers to nonspecific and unintended genetic modifications that can arise through the use of engineered nuclease technologies such as: clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9, transcription activator-like effector nucleases (TALEN), meganucleases, and zinc finger nucleases (ZFN). These tools use different mechanisms to bind a predetermined sequence of DNA (“target”), which they cleave, creating a double-stranded chromosomal break (DSB) that summons the cell's DNA repair mechanisms and leads to site-specific modifications. If these complexes do not bind at the target, often a result of homologous sequences and/or mismatch tolerance, they will cleave off-target DSB and cause non-specific genetic modifications. Specifically, off-target effects consist of unintended point mutations, deletions, insertions inversions, and translocations.

Paula Cannon is a British geneticist and virologist, Distinguished Professor of Molecular Microbiology & Immunology at the University of Southern California. She is a specialist in gene therapy, hematopoietic stem cells, and human immunodeficiency virus (HIV) with particular interest in gene editing and humanized mice.

References

  1. ↑ Deeks, S. G.; McCune, J. M. (2010). "Can HIV be cured with stem cell therapy?". Nature Biotechnology. 28 (8): 807–810. doi:10.1038/nbt0810-807. PMID   20697404. S2CID   20312608.
  2. ↑ Alkhatib, G (2009). "The biology of CCR5 and CXCR4". Current Opinion in HIV and AIDS. 4 (2): 96–103. doi:10.1097/coh.0b013e328324bbec. PMC   2718543 . PMID   19339947.
  3. 1 2 Hütter, G.; Nowak, D.; Mossner, M.; Ganepola, S.; Müßig, A.; Allers, K.; Thiel, E. (2009). "Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation". New England Journal of Medicine. 360 (7): 692–698. doi: 10.1056/nejmoa0802905 . PMID   19213682. S2CID   14905671.
  4. ↑ Carroll, D (2008). "Progress and prospects: zinc-finger nucleases as gene therapy agents". Gene Therapy. 15 (22): 1463–1468. doi:10.1038/gt.2008.145. PMC   2747807 . PMID   18784746.
  5. ↑ Perez, E. E.; Wang, J.; Miller, J. C.; Jouvenot, Y.; Kim, K. A.; Liu, O.; June, C.H. (2008). "Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases". Nature Biotechnology. 26 (7): 808–816. doi:10.1038/nbt1410. PMC   3422503 . PMID   18587387.
  6. ↑ Chung, J.; Rossi, J. J.; Jung, U. (2011). "Current progress and challenges in HIV gene therapy". Future Virology. 6 (11): 1319–1328. doi:10.2217/fvl.11.113. PMC   3383045 . PMID   22754586.
  7. 1 2 Lai, Y. CCR5-targeted hematopoietic stem cell gene approaches for HIV disease: Current progress and future prospects Current Stem Cell Research and Therapy, 2012; 7 (4), pp. 310-317.
  8. ↑ De Silva, E., Stumpf, Michael P.H. (2004). "HIV and the CCR5-D32 Resistance Allele". FEMS Microbiology Letters. 241 (1): 1–12. doi: 10.1016/j.femsle.2004.09.040 . PMID   15556703.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  9. 1 2 3 4 5 6 7 8 Carroll, D (2011). "Genome engineering with zinc-finger nucleases". Genetics. 188 (4): 773–782. doi:10.1534/genetics.111.131433. PMC   3176093 . PMID   21828278.
  10. ↑ Durand, Christine. M, Siliciano, Robert F. (2014). "Dual Zinc-Finger Nucleases Block HIV Infection". Blood. 123 (1): 636–646. doi: 10.1182/blood-2013-11-535542 . PMID   24385492.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  11. 1 2 3 4 5 Urnov, F. D.; Rebar, E. J.; Holmes, M. C.; Zhang, H. S.; Gregory, P. D. (2010). "Genome editing with engineered zinc finger nucleases". Nature Reviews Genetics. 11 (9): 636–646. doi:10.1038/nrg2842. PMID   20717154. S2CID   205484701.
  12. 1 2 Urnov, F. D.; Miller, J. C.; Lee; Beausejour; Rock, J. M.; Augustus, S.; Holmes, M. C. (2005). "Highly efficient endogenous human gene correction using designed zinc-finger nucleases". Nature. 435 (7042): 646–651. Bibcode:2005Natur.435..646U. doi:10.1038/nature03556. PMID   15806097. S2CID   4390010.
  13. ↑ Wayengera, M. "Proviral HIV-genome-wide and pol-gene specific zinc finger nucleases: usability for targeted HIV gene therapy. Theor Biol Med Model, 2011; 8, pp26.
  14. ↑ Wayengera, M.Zinc finger arrays binding human papillomavirus types 16 and 18 genomic DNA: precursors of gene-therapeutics for in-situ reversal of associated cervical neoplasia.Theor Biol Med Model, (2011), 9, pp30.
  15. ↑ Wayengera, M.Identity of zinc finger nucleases with specificity to herpes simplex virus type II genomic DNA: novel HSV-2 vaccine/therapy precursors.Theor Biol Med Model, (2011), 8, pp23.
  16. ↑ Wayengera, M (2003). "HIV and Gene Therapy: The proposed [R-M enzymatic] model for a gene therapy against HIV". Makerere Med J. 38: 28–30.
  17. ↑ Wayengera, M; Kajumbula, H; Byarugaba, W (2007). "Frequency and site mapping of HIV-1/SIVcpz, HIV-2/SIVsmm and Other SIV gene sequence cleavage by various bacteria restriction enzymes: Precursors for a novel HIV inhibitory product". Afr J Biotechnol. 6 (10): 1225–1232.
  18. ↑ Wayengera M, Kajumbula H, Byarugaba W: Identification of restriction endonuclease with potential ability to cleave the HSV-2 genome: inherent potential for biosynthetic versus live microbicides. Theor Biol Med Model. 2008, 5:18.
  19. ↑ Wayengera, M (2008). "Pre-Integration gene slicing (PRINT-GSX) as an alternate or complementary gene therapy modem to RNA interference". J Appl Biol Sci. 1 (2): 56–63.
  20. ↑ Wayengera M: Diverting primary HIV entry and replication to vaginal commensal lactobacillus expressing R-M nucleic enzymatic peptides with potent activity at cleaving proviral DNA as a novel HIV live microbicide strategy. Microbicide- New Delhi, India 2008. Abs-10.
  21. ↑ Wayengera M: Preparing for a Phase 1 Preclinical trial of VRX-SMR: a Lentiviral Vector transduced with restriction enzymes cleaving HIV proviral DNA as a therapeutic vaccine: Opportunities and Challenges. Vaccine Congress -Amsterdam, Netherlands 2007, :24OR.
  22. ↑ Wayengera M: xREPLAB: A recombinant lactobacillus strain producing restriction enzymes with potent activity against HIV proviral DNA as a Live Microbicide Strategy. AIDS vaccine- Washington, Seattle 2007, :P05-01.
  23. ↑ Wayengera, M (2007). "PREX-1979: Modeling the first ever prototype of could be a 5th generation of Microbicides for preventing HIV infection among high-risk women". Afr J Biotechnol. 6 (10): 1221–1224.
  24. 1 2 Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S., & Gregory, P. D. (2010). "Genome Editing with Engineered Zinc Finger Nucleases". Nature Reviews Genetics. 11 (9): 636–646. doi:10.1038/nrg2842. PMID   20717154. S2CID   205484701.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  25. 1 2 3 4 5 6 7 8 9 10 11 Pavletich, N. P.; Pabo, C. O. (1991). "Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A.". Science. 252 (5007): 809–817. Bibcode:1991Sci...252..809P. doi:10.1126/science.2028256. PMID   2028256. S2CID   38000717.
  26. 1 2 3 4 5 6 7 8 9 10 11 Klug, A (2005). "The discovery of zinc fingers and their development for practical applications in gene regulation". Proceedings of the Japan Academy, Series B. 81 (4): 87–102. Bibcode:2005PJAB...81...87K. doi: 10.2183/pjab.81.87 .
  27. ↑ Frankel, A. D.; Berg, J. M.; Pabo, C. O. (1987). "Metal-dependent folding of a single zinc finger from transcription factor IIIA". Proceedings of the National Academy of Sciences. 84 (14): 4841–4845. Bibcode:1987PNAS...84.4841F. doi: 10.1073/pnas.84.14.4841 . PMC   305201 . PMID   3474629.
  28. 1 2 Lee, M. S.; Gippert, G. P.; Soman, K. V.; Case, D. A.; Wright, P. E. (1989). "Three-dimensional solution structure of a single zinc finger DNA-binding domain". Science. 245 (4918): 635–637. Bibcode:1989Sci...245..635L. doi:10.1126/science.2503871. PMID   2503871.
  29. 1 2 3 4 Klug, A.; Schwabe, J. W. (1995). "Protein motifs 5. Zinc fingers". The FASEB Journal. 9 (8): 597–604. doi: 10.1096/fasebj.9.8.7768350 . PMID   7768350. S2CID   24135293.
  30. 1 2 3 4 Stone, D.; Kiem, H. P.; Jerome, K. R. (2013). "Targeted gene disruption to cure HIV". Curr Opin HIV AIDS. 8 (3): 217–23. doi:10.1097/COH.0b013e32835f736c. PMC   4226633 . PMID   23478911.
  31. 1 2 Coakley, E.; Petropoulos, C.J.; Whitcomb, J.M. (2005). "Assessing ch vbgemokine co-receptor usage in HIV". Curr. Opin. Infect. Dis. 18 (1): 9–15. doi:10.1097/00001432-200502000-00003. PMID   15647694. S2CID   30923492.
  32. ↑ Wilen, C.B.; Wang, J.; Tilton, J.C.; et al. (2011). "Engineering HIV-resistant humant CD4+ T cells with CXCR4-specific zinc-finger nucleases". PLOS Pathogens. 7 (4): e1002020. doi: 10.1371/journal.ppat.1002020 . PMC   3077364 . PMID   21533216.
  33. ↑ Didigu, C.A.; Wilen, C.B.; Wang, J. (2013). "Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection". Blood. 123 (1): 61–69. doi:10.1182/blood-2013-08-521229. PMC   3879906 . PMID   24162716.
  34. 1 2 Barton, K. M.; Burch, B. D.; Soriano-Sarabia, N.; Margolis, D. M. (2013). "Prospects for treatment of latent HIV". Clinical Pharmacology and Therapeutics. 93 (1): 46–56. doi:10.1038/clpt.2012.202. PMC   3942883 . PMID   23212106.
  35. 1 2 3 4 5 6 Cathomen, T., & Joung, J. K.. Zinc-finger nucleases: the next generation emerges. Molecular Therapy, (2008) 16(7), pp 1200-1207.
  36. 1 2 Levine, B. L.; Humeau, L. M.; Boyer, J.; MacGregor, R. R.; Rebello, T.; Lu, X.; June, C. H. (2006). "Gene transfer in humans using a conditionally replicating lentiviral vector". Proceedings of the National Academy of Sciences. 103 (46): 17372–17377. Bibcode:2006PNAS..10317372L. doi: 10.1073/pnas.0608138103 . PMC   1635018 . PMID   17090675.
  37. ↑ Varela-Rohena, A.; Carpenito, C.; Perez, E. E.; Richardson, M.; Parry, R. V.; Milone, M.; Riley, J. L. (2008). "Genetic engineering of T cells for adoptive immunotherapy". Immunologic Research. 42 (1–3): 166–181. doi:10.1007/s12026-008-8057-6. PMC   2699549 . PMID   18841331.
  38. 1 2 3 4 5 Rosenberg, T. "The man who had HIV and now does not". New York Magazine. Retrieved January 2013.
  39. ↑ Hütter G, Ganepola S (2011). "Eradication of HIV by transplantation of CCR5-deficient hematopoietic stem cells". The Scientific World Journal . 11: 1068–1076. doi: 10.1100/tsw.2011.102 . PMC   5720062 . PMID   21552772.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.