1. ZFNs: Zinc finger chimeric endonucleases -- a way to make a site-specific double-stranded break at a chosen site Link zinc fingers to a nonspecific endonuclease (a dimer). Use four fingers per endonuclease monomer, so effectively have a 24- basepair recognition site -- long enough to specify unique site in mammalian genomes. Zinc fingers that recognize specific triplets have been identified (most are in proprietary database of a biotech company called Sangamo BioSciences). Use for gene therapy: – Urnov et al. (2005) Nature 435: 646-651 (News and Views, pgs 577-579); Kandavelou et al. (2005) Nature Biotechnology 23: 686-687.

2. Zinc finger endonuclease-mediated gene targeting in human cells Kandavelou et al. (2005) Nature Biotechnology 23: 686-687 Zinc fingers that specifically target particular 3 basepair sequences were developed by phage display and other selection methods. Because they are modular recognition units, they can be linked together in any order to create a reagent that specifically recognizes any desired DNA sequence. Each “N” is a subunit of a dimeric endonuclease.

3. Gene Therapy Treat genetic diseases by correcting gene defect Current gene therapy protocols involve gene addition to insert therapeutic genes randomly in genome using viral vectors -- random insertions can activate oncogenes as seen in gene therapy trials to treat X-SCID. Zinc finger endonuclease-mediated gene targeting uses cell’s homology-directed gene editing process rather than gene addition. Dimerized enzyme introduces double-stranded break near site of mutation. Homologous recombination machinery repairs damage using wild-type gene that was introduced along with zinc finger endonuclease.

4. Gene correction High, 2005, Nature 435; 577-579

5. Life-long supply of anti-HIV neutralizing antibodies Lymphocytes Target HIV Engineering Immunity against HIV David Baltimore, PI, Bill and Melinda Gates Foundation Hematopoietic stem cells (Lentivirus)

6. Clicker question Is it necessary to isolate autologous (a person’s own) hematopoietic stem cells in the Engineering Immunity approach? 1) Yes 2) No

7. Engineering immunity to HIV Hematopoietic stem cell (HSC) transduction with a lentiviral vector (1)Hematopoietic stem cell (HSC) transduction with a lentiviral vector (1) Cell-specific expression (2) Cell-specific expression (2) May be used to express engineered anti-HIV molecules May be used to express engineered anti-HIV molecules Does not rely on host immune system Does not rely on host immune system Not limited to natural antibody architecture Not limited to natural antibody architecture (1)Yang, L., and D. Baltimore, PNAS, 2005. 102: 4518-23. (2)Lois, C., et al., Science, 2002. 295: 868-72.

8. scFv (single-chain Fv) Intact IgG antibody Smaller versions of antibodies against the CCR5 binding site on gp120 neutralize HIV more effectively than intact antibodies, but the immune system only produces intact antibodies These are idealized data from an in vitro neutralization assay.

9. Steric restrictions on antibody access to the coreceptor binding site on gp120 Burton et al. (2005) PNAS 102, 14943-8 scFv and Fab versions of anti-CCR5 antibodies can fit in space between gp120 and target cell membrane. Intact IgG is too big. Explains why scFv and Fabs of anti-CCR5 antibodies neutralize virus better than IgG versions. People don’t normally make scFv and Fab versions of antibodies, but they could be programmed to do so in an Engineering Immunity approach.

10. Antibody design News feature, Nature (2007) volume 446, pp. 964-966

11. Alternative architectures for new anti-HIV molecules Traditional antibody Anthony West, Josh Klein (former Bi1 TA)

12. Single chain bivalent Gly-Ser linker is highly flexibleSingle chain bivalent Gly-Ser linker is highly flexible May favor 1:1 over 2:1 stoichiometry May favor 1:1 over 2:1 stoichiometry Diabodies exhibit restricted torsion angles (1)Diabodies exhibit restricted torsion angles (1) May favor 2:1 over 1:1 stoichiometry May favor 2:1 over 1:1 stoichiometry (1)Lawrence, L.J., A.A. Kortt, et al., FEBS Lett, 1998. 425(3): 479-84. Selection of combining sites may lead to different stoichiometries Different types of bispecific reagents

13. We know very little about how HIV envelope spikes are distributed on virions Zhu et al., 2003, PNAS 100: 15182-15187 Mutant SIV ~75 trimers HIV 7-14 trimers 100 nm Electron tomography of HIV envelope glycoprotein trimers on virions

14. Antibody binding sites on model of trimeric viral spike Burton, Dennis R. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 14943-14948

15. 2G12 Fabs are domain swapped to form a rigid Fab 2 unit. Calarese et al., 2003, Science 300: 2065-71. 2G12 also forms domain-swapped dimers (two IgGs; four Fabs) West et al., 2009, J. Virol. 83: 98-104

16. The dimeric form of 2G12 IgG is more effective in neutralizing HIV Anthony West, Priyanthi Peiris, Joshua Klein Dimer IC 50 : 0.94 ± 0.053 nM Monomer IC 50 : 43 ± 4.9 nM Dimer IC 50 : 0.43 ± 0.066 nM Monomer IC 50 : 30 ± 3.2 nM

17. 2G12 dimer neutralizes more effectively than 2G12 monomer CAVD NAb Core, Beth Huey Tubman, Anthony West, Priyanthi Gnanapragasam

18. Life-long supply of anti-HIV neutralizing antibodies Lymphocytes Target HIV Engineering Immunity against HIV David Baltimore, PI, Bill and Melinda Gates Foundation Hematopoietic stem cells Is it practical to isolate hematopoietic stem cells from every person to be treated?

19. Baltimore/Yang/Wang Approach to Target Recombinant Lentiviruses to Hematopoietic Stem Cells (HSCs) binding induces endocytosis endosomal compartment low pH CD34 Anti-CD34 antibody targets lentivirus to HSCs. Recombinant lentivirus with membrane-bound anti-CD34 plus flu hemagglutinin, which has been mutated so that it no longer binds sialic acids. Receptor-mediated endocytosis brings recombinant lentivirus to an acid endosome. Low pH triggers hemagglutinin to fuse with endosomal membrane. Viral capsid released into cytoplasm of cell.

20. Potentially safer method than lentiviral or retroviral vectors: Use adeno-associated virus (AAV) to deliver genes AAV infects humans and primates – Doesn’t cause disease -- is a satellite virus ssDNA virus, stably integrates into host genome at specific site on chromosome 19 Integrative capacity removed from AAV-based gene therapy vectors. Form episomal (not in a chromosome) concatamers in host cell nucleus – Concatamers retained in non-dividing cells – Concatamers lost during cell division in non-dividing cells T = 1 icosahedral virus Crystal structure described in Xie et al., 2002, PNAS 99 10405-10

21. Long term supply of anti- HIV neutralizing antibodies Engineering Immunity against HIV Gene for anti-HIV NAb or Ab-like protein Engineered recombinant Adeno-Associated Virus Make improved antibody/antibody- like protein reagents starting with known broadly neutralizing anti-HIV antibodies. Persistent muscle cell expression

22. Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys Johnson et al., 2009, Nature Medicine published online 17 May 2009; doi:10.1038/nm.1967 Immunized rhesus macaques with AAV vectors carrying neutralizing antibody constructs Intramuscular injections – achieved µg/ml concentrations in the blood – single injection resulted in long-term (>1 year) expression of biologically-active antibodies Four weeks after intramuscular injection, injected SIV After six more weeks – 6/6 control monkeys infected after challenge. 4/6 developed AIDS after ~one year – 6/9 AAV-injected monkeys were protected against infection, none developed AIDS after ~one year

23. Extra credit opportunity See course website for details Submit an idea for how to prevent or cure HIV infection (due Friday 6/10/11). If you wish to use an “Engineering Immunity” gene therapy approach (as discussed this lecture), follow the instructions on the Bi1 assignments page of the course website for describing your idea. If you have a different sort of idea, follow the guidelines described for a Grand Challenges Exploration grant: go to http://www.gcgh.org/Explorations/Pages/ApplicationInstructions.aspx and and click on "Read Round 7 topics." http://www.gcgh.org/Explorations/Pages/ApplicationInstructions.aspx – The Bill and Melinda Gates Foundation is inviting anyone and everyone to submit short (maximum of two pages) proposals describing new ideas that are “off the beaten track” and “daring in premise.” – Applicants chosen for funding will receive $100,000 to carry out the research. Round 7 submissions were due May 19, 2011, but there will be another round of submissions in Fall 2011. – If anyone is interested in submitting his/her idea for Round 8, we will be happy to discuss it further with you and/or help you write the actual proposal. – If you are chosen for funding, we will find a laboratory at Caltech where you can conduct your research. If you are not chosen for funding, but are committed to trying your idea, we will help you find a way to test your idea in a lab at Caltech or elsewhere as a SURF project.

24. Extra slides

25. 3D reconstruction of SIV virions by cryo-electron tomography Zanetti, G., et al., PLoS Pathogens 2, e83 (2006) Trimeric HIV envelope spike structures have been examined on viruses using cryoelectron tomography The HIV envelope spike is a trimer of gp120/gp41* heterodimers. *gp (glycoprotein) 120 is 120 kDa; gp41 is 41 kDa.

26. Zanetti, G., et al., PLoS Pathogens 2, e83 (2006) Zhu, P., et al., Nature 441, 847 (2006) The resulting structure is low resolution, but shows that the trimer should be accessible to antibodies

27. Clicker question: Companies have spent a lot of money trying to develop a soluble form of CD4 that could be injected into an HIV-positive patient. The reasoning behind this approach is that… (a) Soluble CD4 would compete with membrane bound CD4 to prevent gp120 from binding T cells. (b) Injected soluble CD4 is unlikely to trigger an immune reaction. (c) gp120 can’t mutate its CD4 binding site without reducing its ability to infect T cells. (d) All of the above. (e) None of the above. CCR5 gp120 CD4

28. Clicker question: Companies have spent a lot of money trying to develop a soluble form of CD4 that could be injected into an HIV-positive patient. The reasoning behind this approach is that… (a) Soluble CD4 would compete with membrane bound CD4 to prevent gp120 from binding T cells. (b) Injected soluble CD4 is unlikely to trigger an immune reaction against it. (c) gp120 can’t mutate its CD4 binding site without reducing its ability to infect T cells. (d) All of the above. (e) None of the above. soluble CD4

29. Ping Zhu, Ken Roux, Univ. Florida