# Precision Apheresis: stem cell isolation from patients with sickle cell disease for gene therapy using high-throughput microfluidics

> **NIH NIH K25** · MASSACHUSETTS GENERAL HOSPITAL · 2024 · $137,183

## Abstract

PROJECT SUMMARY
 Sickle Cell Disease (SCD) affects millions of people around the world. Recently, stem cell gene therapy has
emerged as a potentially curative option for SCD. Obtaining a sufficient dose of hematopoietic stem and
progenitor cells (HSPCs) from peripheral blood is paramount to the success of these gene therapies. However,
the higher numbers of RBCs in apheresis products can adversely impact the yield of valuable hematopoietic
stem cells during purification (~46% loss). There is, therefore, an immediate unmet need to develop an isolation
technology that can efficiently recover hematopoietic stem cells from apheresis products, irrespective of their
hematocrits. To address this challenge, I will develop a microfluidic HSPC isolation chip (HSPC-iChip) capable
of recovering >95% CD34+ cells from full apheresis products (~300 mL) in an hour (Aim 1). I will bring
advancements (in microfluidic technologies) from the field of cancer diagnostics to the field of
hematology to accomplish this. My central hypothesis is that the HSPC-iChip can isolate highly viable and
functional hematopoietic stem cells. To test this hypothesis, I will genetically edit the isolated stem cells and
analyze engraftment, disease correction, and human hematopoiesis in NBSGW mice (Aim 1). Additionally, under
the influence of centrifugal forces, the hypercoagulable state of sickle cell patients can lead to the formation of
cell clusters. These clusters have been observed to destabilize the cell collection interface, requiring highly
skilled apheresis operators for stem cell collection from sickle cell patients. Once the apheresis product is
collected, subsequent purification of ~1% HSPCs from the rest of the cells further necessitates specialized
instruments and consumables. This restricts a broader implementation of sickle cell gene therapy as most
patients reside in low-resource settings where skilled labor, bio-cleanrooms, and financial capabilities
are restricted. To address this challenge, in Aim 2, I will test the feasibility of a Precision Apheresis
technology that can directly separate HSPCs from peripheral circulation in a single step based on their
surface epitopes (CD34). The training objective of this project is to provide Dr. Mishra—who has a strong
background in microfluidics and cell sorting—with additional scientific training from leading pioneers in
therapeutic gene editing for hemoglobinopathies (Dr. Bauer, Boston Children's Hospital/Harvard), stem cell
apheresis and pathology (Dr. Manis, Boston Children's Hospital/Harvard), clinical hematology (Dr. Azar,
MGH/Harvard) high-throughput microfluidics (Dr. Toner, lead mentor, MGH/Harvard), animal models and
advanced tissue culture (Dr. Haber, co-mentor, MGH/Harvard), nanoparticle kinetics (Dr. Bhatia, MIT),
computational modeling of blood cells (Dr. Koumoutsakos, Harvard), and closed-loop mouse-chip models (Dr.
Manalis, MIT). This additional training will prepare Dr. Mishra to lead an independent transdis...

## Key facts

- **NIH application ID:** 10899569
- **Project number:** 5K25HL169816-02
- **Recipient organization:** MASSACHUSETTS GENERAL HOSPITAL
- **Principal Investigator:** Avanish Mishra
- **Activity code:** K25 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $137,183
- **Award type:** 5
- **Project period:** 2023-08-05 → 2028-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10899569

## Citation

> US National Institutes of Health, RePORTER application 10899569, Precision Apheresis: stem cell isolation from patients with sickle cell disease for gene therapy using high-throughput microfluidics (5K25HL169816-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10899569. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*