Individual Final Project

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Autologous engraftment of immunoengineered hematopoietic stem cells for Env-targetting broad neutralizing antibodies

SECTION 1: Abstract

Generating humoral immunity against HIV, particularly the difficulty of eliciting broadly neutralizing antibodies (bnAbs) through conventional vaccination remains a challenge. Current approaches rely on complex immune maturation pathways that are inefficient and often impaired in immunocompromised individuals, highlighting the need for alternative strategies. The objective here is to develop a stem cell–based immunoengineering platform capable of producing long-term, self-renewing humoral immunity by genetically programming antibody specificity. The hypothesis here is that autologous hematopoietic stem cells engineered to carry predefined anti-HIV Env bnAb heavy and light chain genes will differentiate into B-cell lineages that express functional bnAb receptors, then produce the antibodies that are predefined. The first milestone will consist of the design of a construct encoding bnAb heavy and light-chain sequences, which can be gathered from scientific literature, secondly, perform CRISPR-mediated genome editing in human CD34+ hematopoietic stem and progenitor cells, and third, evaluate differentiation into B-cell progeny and expression of the engineered receptor.

Methods will include ex vivo isolation of CD34+ HSPCs from peripheral blood, CRISPR-Cas9–based genome editing, short-term stem cell culture for HSPCs, and validation through molecular assays, sequencing, and flow cytometry to confirm integration and antigen-specific receptor expression.

SECTION 2: Project Aims

Aim 1: Experimental Aim

The first aim of my final project is to genetically engineer autologous hematopoietic stem cells to carry predefined anti-HIV Env broadly neutralizing antibody heavy and light-chain genes, so that their B-cell progeny express an HIV-specific broadly neutralizing B-cell receptor, by utilizing CRISPR-based gene editing, a DNA design for bnAb heavy and light chain insertion, ex vivo HSC culture, and differentiation and molecular validation assays.

Aim 2: Development Aim

This could shift prevention and treatment from repeated drug administration to a one-time or very infrequent treatment.

Aim 3: Visionary Aim

If fully realized, this approach would challenge the current paradigm of lifelong antiretroviral therapy by overcoming the challenge that viruses like HIV, denguevirus, and influenza has: mutations and serotypes. Essentially, this could be a universal treatment if done for each virus that has these kind of challenges.

SECTION 3: Background

Briefly summarize two peer-reviewed research citations relevant to your research (minimum four sentences).

A very relevant study by Jardine et al., (2016) shows that immunogens like eOD-GT8 can activate rare VRC01-class precursor B cells and recover their paired heavy and light chain sequences, but these cells are extremely rare and require complex maturation.

And another one, by Porteus et al., (2026), which demonstrates that CRISPR-edited HSPCs can be genetically engineered to produce B cells that secrete functional antibodies.

Explain how your project is novel or innovative. (Minimum 3 sentences.)

This is innovative because it applies CRISPR-based genome engineering of hematopoietic stem cells to directly program the production of HIV broadly neutralizing antibodies, rather than relying on immunogems to elicit them. It introduces a new approach that assures the production of known bnAbs that are known to neutralize HIV virions. This challenges the existing belief that protective immunity must be induced through natural immune processes.

SECTION 4: Experimental design, techniques, tools and technology

Please identify at least one (ideally many) aspect(s) of your project that you will measure. It could be the mass or sequence of a protein, the presence, absence, or quantity of a biomarker, etc.

  • The integration of the VRC01 sequence in the hematopoietic cells.

Please describe all of the elements you would like to measure, and furthermore describe how you will perform these measurements.

  • VRC01 sequence (bnAbs that target Env) in the hematopoietic cells
  • A GFP reporter gene

What are the technologies you will use (e.g., gel electrophoresis, DNA sequencing, mass spectrometry, etc.)? Describe in detail.

  • Flow cytometry we can get to know how many cells have been genetically modified.
  • PCR can be done too, targetting the construct we’ve inserted with primers designed just for that.

Sequence to be inserted:

3ngbE|Fab VRC01, anti-gp120 CD4-binding site (CD4bs) [HIV-1], broadly neutralizing|||VH-CH1 (VH (1-121) [D1] + CH1 (123-218) [D2])|||||||224|224|||MW undefined|MW undefined| QVQLVQSGGQMKKPGESMRISCRASGYEFIDCTLNWIRLAPGKRPEWMGWLKPRGGAVNY ARPLQGRVTMTRDVYSDTAFLELRSLTVDDTAVYFCTRGKNCDYNWDFEHWGRGTPVIVS SPSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSC reverse translation of sample sequence to a 672 base sequence of most likely codons. caggtgcagctggtgcagagcggcggccagatgaaaaaaccgggcgaaagcatgcgcatt agctgccgcgcgagcggctatgaatttattgattgcaccctgaactggattcgcctggcg ccgggcaaacgcccggaatggatgggctggctgaaaccgcgcggcggcgcggtgaactat gcgcgcccgctgcagggccgcgtgaccatgacccgcgatgtgtatagcgataccgcgttt ctggaactgcgcagcctgaccgtggatgataccgcggtgtatttttgcacccgcggcaaa aactgcgattataactgggattttgaacattggggccgcggcaccccggtgattgtgagc agcccgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagc ggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtg agctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagc agcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccag acctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagcggaa ccgaaaagctgc

Bibliographic references

Jardine, J. G., Kulp, D. W., Havenar-Daughton, C., Sarkar, A., Briney, B., Sok, D., Sesterhenn, F., Ereño-Orbea, J., Kalyuzhniy, O., Deresa, I., Hu, X., Spencer, S., Jones, M., Georgeson, E., Adachi, Y., Kubitz, M., deCamp, A. C., Julien, J. P., Wilson, I. A., Burton, D. R., … Schief, W. R. (2016). HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen. Science (New York, N.Y.), 351(6280), 1458–1463.

Porteus, M., Luna, S., Feist, W., Utz, A., Afaghani, J., Miyauchi, M., … & Schmiderer, L. (2026). Engineered hematopoietic stem cells give rise to therapeutic antibody secreting B cells. https://www.researchsquare.com/article/rs-9269825/v1

Wu, X., Yang, Z. Y., Li, Y., Hogerkorp, C. M., Schief, W. R., Seaman, M. S., Zhou, T., Schmidt, S. D., Wu, L., Xu, L., Longo, N. S., McKee, K., O’Dell, S., Louder, M. K., Wycuff, D. L., Feng, Y., Nason, M., Doria-Rose, N., Connors, M., Kwong, P. D., … Mascola, J. R. (2010). Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science (New York, N.Y.), 329(5993), 856–861. https://doi.org/10.1126/science.1187659

Sequences and their origin

CAS9_STRP1 (https://www.uniprot.org/uniprotkb/Q99ZW2/entry#sequences): atggataaaaaatatagcattggcctggatattggcaccaacagcgtgggctgggcggtg attaccgatgaatataaagtgccgagcaaaaaatttaaagtgctgggcaacaccgatcgc catagcattaaaaaaaacctgattggcgcgctgctgtttgatagcggcgaaaccgcggaa gcgacccgcctgaaacgcaccgcgcgccgccgctatacccgccgcaaaaaccgcatttgc tatctgcaggaaatttttagcaacgaaatggcgaaagtggatgatagcttttttcatcgc ctggaagaaagctttctggtggaagaagataaaaaacatgaacgccatccgatttttggc aacattgtggatgaagtggcgtatcatgaaaaatatccgaccatttatcatctgcgcaaa aaactggtggatagcaccgataaagcggatctgcgcctgatttatctggcgctggcgcat atgattaaatttcgcggccattttctgattgaaggcgatctgaacccggataacagcgat gtggataaactgtttattcagctggtgcagacctataaccagctgtttgaagaaaacccg attaacgcgagcggcgtggatgcgaaagcgattctgagcgcgcgcctgagcaaaagccgc cgcctggaaaacctgattgcgcagctgccgggcgaaaaaaaaaacggcctgtttggcaac ctgattgcgctgagcctgggcctgaccccgaactttaaaagcaactttgatctggcggaa gatgcgaaactgcagctgagcaaagatacctatgatgatgatctggataacctgctggcg cagattggcgatcagtatgcggatctgtttctggcggcgaaaaacctgagcgatgcgatt ctgctgagcgatattctgcgcgtgaacaccgaaattaccaaagcgccgctgagcgcgagc atgattaaacgctatgatgaacatcatcaggatctgaccctgctgaaagcgctggtgcgc cagcagctgccggaaaaatataaagaaattttttttgatcagagcaaaaacggctatgcg ggctatattgatggcggcgcgagccaggaagaattttataaatttattaaaccgattctg gaaaaaatggatggcaccgaagaactgctggtgaaactgaaccgcgaagatctgctgcgc aaacagcgcacctttgataacggcagcattccgcatcagattcatctgggcgaactgcat gcgattctgcgccgccaggaagatttttatccgtttctgaaagataaccgcgaaaaaatt gaaaaaattctgacctttcgcattccgtattatgtgggcccgctggcgcgcggcaacagc cgctttgcgtggatgacccgcaaaagcgaagaaaccattaccccgtggaactttgaagaa gtggtggataaaggcgcgagcgcgcagagctttattgaacgcatgaccaactttgataaa aacctgccgaacgaaaaagtgctgccgaaacatagcctgctgtatgaatattttaccgtg tataacgaactgaccaaagtgaaatatgtgaccgaaggcatgcgcaaaccggcgtttctg agcggcgaacagaaaaaagcgattgtggatctgctgtttaaaaccaaccgcaaagtgacc gtgaaacagctgaaagaagattattttaaaaaaattgaatgctttgatagcgtggaaatt agcggcgtggaagatcgctttaacgcgagcctgggcacctatcatgatctgctgaaaatt attaaagataaagattttctggataacgaagaaaacgaagatattctggaagatattgtg ctgaccctgaccctgtttgaagatcgcgaaatgattgaagaacgcctgaaaacctatgcg catctgtttgatgataaagtgatgaaacagctgaaacgccgccgctataccggctggggc cgcctgagccgcaaactgattaacggcattcgcgataaacagagcggcaaaaccattctg gattttctgaaaagcgatggctttgcgaaccgcaactttatgcagctgattcatgatgat agcctgacctttaaagaagatattcagaaagcgcaggtgagcggccagggcgatagcctg catgaacatattgcgaacctggcgggcagcccggcgattaaaaaaggcattctgcagacc gtgaaagtggtggatgaactggtgaaagtgatgggccgccataaaccggaaaacattgtg attgaaatggcgcgcgaaaaccagaccacccagaaaggccagaaaaacagccgcgaacgc atgaaacgcattgaagaaggcattaaagaactgggcagccagattctgaaagaacatccg gtggaaaacacccagctgcagaacgaaaaactgtatctgtattatctgcagaacggccgc gatatgtatgtggatcaggaactggatattaaccgcctgagcgattatgatgtggatcat attgtgccgcagagctttctgaaagatgatagcattgataacaaagtgctgacccgcagc gataaaaaccgcggcaaaagcgataacgtgccgagcgaagaagtggtgaaaaaaatgaaa aactattggcgccagctgctgaacgcgaaactgattacccagcgcaaatttgataacctg accaaagcggaacgcggcggcctgagcgaactggataaagcgggctttattaaacgccag ctggtggaaacccgccagattaccaaacatgtggcgcagattctggatagccgcatgaac accaaatatgatgaaaacgataaactgattcgcgaagtgaaagtgattaccctgaaaagc aaactggtgagcgattttcgcaaagattttcagttttataaagtgcgcgaaattaacaac tatcatcatgcgcatgatgcgtatctgaacgcggtggtgggcaccgcgctgattaaaaaa tatccgaaactggaaagcgaatttgtgtatggcgattataaagtgtatgatgtgcgcaaa atgattgcgaaaagcgaacaggaaattggcaaagcgaccgcgaaatattttttttatagc aacattatgaacttttttaaaaccgaaattaccctggcgaacggcgaaattcgcaaacgc ccgctgattgaaaccaacggcgaaaccggcgaaattgtgtgggataaaggccgcgatttt gcgaccgtgcgcaaagtgctgagcatgccgcaggtgaacattgtgaaaaaaaccgaagtg cagaccggcggctttagcaaagaaagcattctgccgaaacgcaacagcgataaactgatt gcgcgcaaaaaagattgggatccgaaaaaatatggcggctttgatagcccgaccgtggcg tatagcgtgctggtggtggcgaaagtggaaaaaggcaaaagcaaaaaactgaaaagcgtg aaagaactgctgggcattaccattatggaacgcagcagctttgaaaaaaacccgattgat tttctggaagcgaaaggctataaagaagtgaaaaaagatctgattattaaactgccgaaa tatagcctgtttgaactggaaaacggccgcaaacgcatgctggcgagcgcgggcgaactg cagaaaggcaacgaactggcgctgccgagcaaatatgtgaactttctgtatctggcgagc cattatgaaaaactgaaaggcagcccggaagataacgaacagaaacagctgtttgtggaa cagcataaacattatctggatgaaattattgaacagattagcgaatttagcaaacgcgtg attctggcggatgcgaacctggataaagtgctgagcgcgtataacaaacatcgcgataaa ccgattcgcgaacaggcggaaaacattattcatctgtttaccctgaccaacctgggcgcg ccggcggcgtttaaatattttgataccaccattgatcgcaaacgctataccagcaccaaa gaagtgctggatgcgaccctgattcatcagagcattaccggcctgtatgaaacccgcatt gatctgagccagctgggcggcgat

CMV Enhancer (https://benchling.com/benchling/f/lib_iuOubVW42Y-promoter-sequences/seq_pY99leaE-cmv-promoter/edit): CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG

CMV Promoter (https://benchling.com/benchling/f/lib_iuOubVW42Y-promoter-sequences/seq_pY99leaE-cmv-promoter/edit): GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT

U6 Promoter (https://www.addgene.org/browse/sequence/135959/) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacacc