1. 1 Genome Editing-Based HIV Therapies Rehana Kousar PhD Scholar 06-arid-746 Department of Botany

2. Trends in Biotechnology Review Genome Editing-Based HIV Therapies Wang-Gang Gu15 January 2015 Genome editing (GE)-based HIV therapy is achieved by modification of infection-related genes to produce HIV- resistant cells followed by reinfusion of the modified cells into patients. The ultimate goal is to achieve a functional or actual cure for HIV infection. Despite multiple potential targets for GE-based HIV therapies, CCR5 is the most feasible owing to the naturally existing CCR5 δ32 genotype which confers resistance to HIV. A recent clinical trial of infusion of modified autologous CD4 + T cells proved safety and efficacy within the limits of the studies. However, long-term evaluation of the safety and efficacy is required before GE-based HIV therapy is ready for clinical implementation. 2

3. Contents HIV HIV Infection Clinical treatment of AIDS GE-based Therapies Advances in GE-based Therapies Targets of GE-based Therapies Concerns in GE-based Therapies Conclusions Future perspective 3

4. HIV The Human Immunodeficiency Virus (HIV) is a lentivirus that causes AIDS i-e., acquired immunodeficiency syndrome A serious infectious disease characterized by the systemic collapse of the immune system that allows life-threatening opportunistic infections and cancers to thrive. The pathogen was identified 2 years after the first report of the disease in 1981. 4

5. HIV Infection It infects vital cells in the human immune system such as CD4+ T lymphocytes, Peripheral blood mononuclear cells (PBMC), Monocytes, Dendritic cells, Macrophages 5

6. HIV infection leads to low levels of CD4 + T cells through a number of mechanisms, Apoptosis of uninfected bystander cells, direct viral killing of infected cells, killing of infected CD4 + T cells by CD8 cytotoxic lymphocytes When CD4 + T cell numbers decline below a critical level, cell-mediated immunity is lost, and the body becomes progressively more susceptible to opportunistic infections. 6

7. Types of HIV Two types of HIV have been characterized: HIV-1 and HIV-2. HIV-1 is the virus that was initially discovered is more virulent, more infective, and is the cause of the majority of HIV infections globally. SpeciesVirulenceInfectivityPrevalence Inferred origin HIV-1High Global Common Chimpanzee HIV-2LowerLowWest Africa Sooty Mangabey 7

8. HIV Genome The HIV-1 genome is composed of two identical copies of single-stranded RNA, consists of nine genes, The high mutation rate during the replication cycle results in a great likelihood for the development of drug resistance of the virus. 8

9. HIV Genome 9

10. 10

11. Clinical Treatment of AIDS More than 30 drugs have been developed for the clinical treatment of AIDS. No effective treatment to eliminate the virus from patients Preventive vaccine for HIV would be ideal, developing a successful preventive vaccine for HIV is a long and complicated process. 11

12. Antiretroviral medicines Nucleoside/nucleotide reverse transcriptase inhibitors Non-nucleoside reverse transcriptase inhibitors (NNRTIs), Protease inhibitors (PIs), Entry inhibitors, Integrase inhibitors 12

13. Targets of HIV Therapies Multiple proteins involved in the HIV life cycle have been validated for antiviral drug development. Theoretically, each step of HIV replication could be targeted for anti-HIV therapy. The HIV-1 genome consists of nine genes, which are also important targets for anti-HIV research. 13

14. HAART Highly-Active Antiretroviral Therapy The only effective therapy used in clinical treatment for HIV infection A combination of antiviral drugs are used targeting different steps in the virus life cycle. It is used to slow the rate at which HIV makes copies of itself (multiplies) in the body 14

15. Why GE-based Therapies An increasing number of patients have to face the problem that, due to the serious drug resistance, there are no suitable drugs to choose from, for use in HAART for HIV infection. 15

16. GE-based Therapies Genome editing (GE)-based HIV therapy is Modification of infection-related genes to produce HIV resistant cells followed by reinfusion of the modified cells into patients. The ultimate goal is to achieve a functional or actual cure for HIV infection. 16

17. In 2009, a patient was functionally cured of HIV infection by transplantation of allogeneic stem cells from a donor homozygous for the CCR5 δ32 allele. There is a small, but reasonable chance of finding an human leukocyte antigen HLA-matched donor homozygous for the CCR5 δ32 allele. Mimicking the natural homozygous CCR5 δ32 by using GE technologies to induce the mutation is a viable choice. 17

18. The success of the stem cell transplant has encouraged the continued study of GE-based HIV therapies. The general concerns of GE-based HIV therapies include target selection, the choice of GE technology, and evaluation of safety and efficacy. 18

19. Advances in GE-based Therapies Transcription activator-like effector nucleases (TALENs) Zinc-finger nucleases (ZFNs) Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 systems The ZFN system--most widely used, there are only a few reports of GE-based HIV therapies using the TALEN and CRISPR/Cas 9 systems, these two GE tools may have great potential. 19

20. ZFNs: Zinc-finger nuclease Origin: Eukaryotes Compatible with various viral vectors; each ZF module (30 amino acids) recognizes three sequence-specific nucleotides; widely used in various cell types and multiple organisms including humans 20

21. TALENs: Transcription activator-like effector nuclease Origin: Xanthomonas TALEs consist of multiple 33–35 amino acid repeats in which each repeat recognizes only one nucleotide. TALENs result in targeted double-stranded breaks which activate DNA damage response pathways. 21

22. CRISPR: Clustered regularly interspaced palindromic repeats Origin: Bacteria DNA loci contained in bacterial genomes. consists of an array composed of 21–47 nucleotide (32 on average) short direct repeats with various intervening spacers. is one component of the pathway used by bacteria to destroy foreign invaders. RNA-guided DNA endonuclease; targets multiple sites simultaneously to produce large gene fragment deletions. 22

23. The major strategy for GE-based HIV therapies is to produce engineered immune cells that are resistant to HIV infection or replication. The possible cell types for modification are hematopoietic stem cells dendritic cells and CD4+ T cells CD4+ T cells and CD34+ stem cells are the two groups of cells, most frequently used for GE- based HIV therapies. 23

24. Method of GE-based Therapy 24

25. Approaches of GE-based Therapies The current approaches are mainly classified into two categories: Therapies targeting host genes involved in virus infection and replication, such as CCR5 Therapies introducing genes that interfere with HIV replication, such as host restriction factors and fusion inhibitors 25

26. Targets of GE-based Therapies CD4, the major receptor for HIV, is very important for the human immune system, Deletion of the CD4 gene is lethal. Several clinical trials involving CD4 modification for HIV therapies have been conducted, but these studies are no longer being investigated for clinical treatment. The CD4 receptor may not be an appropriate target, the co-receptors for viral entry are ideal targets for disruption. 26

27. Targets CCR5 CXCR4 Proviral DNA Other targets  Lens epithelium-derived growth factor (LEDGF)  The mitochondrial translocator protein (TSPO) 27

28. CCR5 C-C chemokine receptor type 5 A naturally existing 32 bp deletion in the CCR5 gene leads to a non-functional receptor, which confers resistance to HIV strains that use CCR5. Cells that are naturally resistant to HIV are usually from donors with the CCR5 d32 genotype Multiple methods have been developed to construct engineered cells with non-functional CCR5 28

29. ZFN - mediated modification of CCR5 ZFN-mediated modification of CCR5 induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) demonstrated the potential for treatment of HIV infection Mice treated with CCR5-disrupted hematopoietic stem or progenitor cells (HSPCs) achieved resistance to HIV infection Permanent dysfunction of CCR5 by modification with ZFN 29

30. Continued … In a study, 12 patients with chronic aviremic HIV infection received a single dose of autologous CD4 T cells modified by ZFN. Immune reconstitution and HIV resistance were evaluated by CD4 T cell count and viral RNA or DNA detection. A significant increase in the CD4 T cell count was observed and blood HIV DNA levels decreased in most patients. 30

31. CRISPR/Cas9: mediated modification of CCR5 Recently, hiPSCs homozygous for the CCR5 d32 mutation were produced using TALEN or CRISPR/Cas9 system in combination with piggyBac technology These engineered cells showed resistance to HIV-1 challenge when differentiated into monocytes/macrophages. 31

32. CXCR4: C-X-C chemokine receptor type 4 Disruption of the C-X-C chemokine receptor type 4 gene (CXCR4) with ZFN cause resistance to T-tropic HIV in several studies Human T cells resistance to X4-tropic HIV-1 strains. also conferred resistance to X4 HIV-1 in humanized mouse. 32

33. Continued … In another study, disruption of CXCR4 with ZFNs in CD4+ T cells provided protection from HIV-1 infection in tissue culture and in mice. After engraftment of engineered T cells, HIV- 1-infected mice had lower viral load. A safe mode of disruption of CXCR4 is still needed to be discovered. 33

34. Proviral DNA Eliminating the HIV proviral DNA from infected cells with GE technologies The incurability of HIV infection can be attributed to the integration of viral DNA into the human genome and the persistence of proviral DNA. 34

35. Cleavage by ZFNs Up to 80% of integrated viral DNA can be removed from the human genome with ZFNs targeting the proviral HIV Pol gene or the whole proviral genome Three ZFNs were designed to cleave the conserved regions of Gag, Pol, and Rev The full-length proviral DNA can also be excised from human genome with ZFNs targeting conserved regions within HIV-1 50 and 30 long terminal repeats (LTRs). 35

36. Cleavage by CRISPR/Cas9 The LTRs target sites were efficiently cleaved and mutated by the system. The CRISPR/Cas9 system was also able to delete internal HIV genes from a human chromosome Highly specific targets were identified and efficiently edited by Cas9 system with guide RNAs (gRNA) homologous to sequences within the LTR U3 region of HIV-1 36

37. HIV-1 infection was prevented in the presence of multiplex gRNAs in cells expressing Cas9. ZFNs have also been designed to target proviral DNA of other viruses including HTLV-1, HSV-2 and HBV Major challenge in clinical treatment is how to deliver the ZFNs or CRISPR/Cas9 system to the latently infected cells accurately because these cells are extremely rare. 37

38. LEDGF Lens Epithelium-derived Growth Factor The first well-characterized cellular cofactor for HIV-1 integrase. It plays a crucial role in viral integration, tethering the pre-integration complex (PIC) to the nucleus through interaction with integrase. Similarly to the case of CCR5, in CD4+ T cells, LEDGF depletion is well tolerated. 38

39. Continued … In recent studies, all LEDGF expression was completely eradicated with TALENs. The deletion of whole LEDGF gene or the exons encoding the integrase binding domain (IBD) resulted in inhibition of viral integration and impaired virus replication in human cell lines. 39

40. TSPO: Mitochondrial Translocator Protein was found to inhibit HIV-1 Env glycoprotein expression Although the CRISPR/Cas9 system was only used to generate a TSPO knockout in 293T cells for this study, the locus could be an attractive target for other GE-based strategies 40

41. Co-receptor Usage Shift Co-receptor usage shift is an important problem in anti-HIV research. CCR5 serves as the predominant co-receptor in the early stage of HIV infection, and CXCR4 plays a key role as co-receptor when stable infection is established. HIV co-receptor usage can switch between CCR5 and CXCR4, resulting in failure of protection if therapies target only one of these co-receptors. 41

42. 42

43. Concerns in GE-based Therapies Several issues give cause for concern. Safety: is a fundamental concern in GE-based therapies. A long-term safety evaluation is needed. Efficacy: It has yet to be determined if the goal of the therapy will be to cure the HIV infection or control the progression of AIDS 43

44. A technical concern for GE tools is off-target cleavage events, which can lead to unintended disruption of physiologically important genes, and can have unpredictable consequences. Predicting possible off-target cleavage sites and understanding their effects will greatly improve the efficiency and the safety of GE- based therapies. 44

45. Conclusion The current GE-based HIV therapies are mainly based on reinfusion of modified CD4+ T lymphocytes or CD34+ stem cells. CCR5 is the major current focus because naturally existing CCR5 d32 confers resistance to HIV infection, Production of HIV-resistant cells with an artificial CCR5 mutation and reinfusing the cells into patients to confer HIV resistance is the most common strategy. 45

46. Future Perspective Several important scientific problems remain to be addressed Co-receptor Usage Shift Off-target events The molecular mechanism of the functional cure is not yet clear. With the development of GE technologies and better understanding of the HIV infection process, more targets may be approached. 46

47. Reference: Gang W.G., 2015. Genome editing-based HIV therapies. TIBTEC-1229: 1-8 47

48. 48