Matt Gethers/20.380 HIV Project/Technical Paper/Matt Design
Stem Cell Collection
- How to collect/purify RBC progenitors?
- Are there easy kit protocols to do this?
- What are concentrations of progenitors in blood?
- How many cells do you need to engineer?
- How do you store blood and cells during the engineering?
- Will samples be engineered in the field or sent back to lab facilities?
- Keeping samples for later use/creation of a library?
- Need storage facilities and permission from patients.
- Could these collected progenitors be infected?
- Possible to cure them?
- Need to express CD4+ and Chemokine co-receptors on surface
- Maybe express library of receptors, or create library of blood cells with different receptors.
- What will genetic construct look like? Separate operons for everything? Same operon? Polycistronic? Post-transcriptional/translational regulation to further refine time expression profile?
- What's vector?
- How transfecting?
- Need to express RNAses
- Post-transcriptional/translational regulation to refine expression profile?
- Protection of progenitor from infection through siRNA.
Reintroduction of Progenitors to Patient
- Paper to demonstrate that reintroduction of ex vivo cells works.
- How many cells need to be introduced to the patient in order to establish necessary presence?
- How to deliver?
- Injection? Into where?
- Keep samples of the engineered cells in labs?
- Need infrastructure and permission from patients.
- Expand and differentiate cells ex vivo to test before introducing to patient?
- Need to test every batch of cells before giving to patient.
- Need FDA blanket approval?
Differentiation in vivo
- When/how will progenitors be induced to differentiate?
- Expression profile of genes
- Modeling of transcription specifically with respect to RNAse expression
- Need alternate regulation mechanisms to refine time expression profile?
- Differentiation kinetics, resultant population of progenitors, sink-RBCs.
- Virions bind RBCs, get caught in trap.
- What ratio of RBCs to T-cells/virions is necessary for viral ablation?
- Model birth, death, infection of RBCs, T-cells, HIV.
- Is appropriate metric surface area and receptor density?
- Concerns about selecting for anti-trap HIV?
- Possible to have RBCs evolve with virus through some sort of progenitor sensor system?
- Drug supersensitivity?
- Dying, take it away.