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.
Hypothesis: Substitution of a bacteriophage’s replisome with an orthogonal T7 replisome for continuous hypermutation directed towards stability The idea of a proposal comes from an article by Diercks et al., 2024, in which they use a very faulty replisome that induces hypermutation, in which they direct towards a very high antibiotic resistance.
Subsections of Projects
Individual Final Project
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
Hypothesis: Substitution of a bacteriophage’s replisome with an orthogonal T7 replisome for continuous hypermutation directed towards stability
The idea of a proposal comes from an article by Diercks et al., 2024, in which they use a very faulty replisome that induces hypermutation, in which they direct towards a very high antibiotic resistance.
Choose one or two main goals from the list that you think you can address computationally (e.g., “We’ll try to stabilize the lysis protein,” or “We’ll attempt to disrupt its interaction with E. coli DnaJ.”).
Goal #1: We’ll try to make the bacteriophage more resistant to varying environmental conditions such as temperature and pH.
Goal #2: We’ll try to reach this stability without compromising the bacteriophage’s infectivity
Write a 1-page proposal (bullet points or short paragraphs) describing:
Which tools/approaches from recitation you propose using (e.g., “Use Protein Language Models to do in silico mutagenesis, then AlphaFold-Multimer to check complexes.”).
Why do you think those tools might help solve your chosen sub-problem?
Name one or two potential pitfalls (e.g., “We lack enough training data on phage–bacteria interactions.”).
Include a schematic of your pipeline.
Proposal
My proposal consists on a bacteriophage based on the T7 replisome, since T7 encodes much of its own replication machinery, a lower-fidelity replisome, we can generate phages that are both faulty, but also excel at our characteristic of interest: stability. By exposing that population to a defined stress linked to stability, and then isolate the phages that remain infectious using plaque-based assays, we can recover viable survivors. After sequencing several independently recovered survivor phages, I would compare their encoded proteins with Clustal Omega, from that, we could build a consensus-style view of which residues remain strongly conserved and which sites may repeatedly change under selection, and so, we can make a construct of, hopefully, a very stable bacteriophage
Two potential pitfalls could be
Results for clustal-omega could be too divergent, making it difficult to decide on conserved residues
There could be no stable bacteriophages due to the faultiness of the replisome
Results for clustal-omega
(We’re doing a theoretical alignment, given that we cannot perform the proposed experiment to carry on real alignments)
Schematic
Bibliographic references
Diercks, C. S., Sondermann, P. J., Rong, C., Dik, D. A., Gillis, T. G., Ban, Y., & Schultz, P. G. (2024). An Orthogonal T7 Replisome for Continuous Hypermutation and Accelerated Evolution in E. coli. bioRxiv : the preprint server for biology, 2024.07.25.605042. https://doi.org/10.1101/2024.07.25.605042
