Mara Pavel-Dinu: AABB Foundation Supports HSCT-Extending Research

Pavel-Dinu's project focused on understanding how the genetic engineering process impacts HSCs' regenerative capacity. In particular, she investigates HSCs’ metabolic adaptations and cellular stress responses to the ex vivo genome editing (GE) process under near-physiological culture conditions that mimic the bone marrow environment. The hope is that through this work, it may be possible to repair the genetic defects of patient-derived HSCs and also to restore the cells’ fitness before transplanting into patients. This emerging gene and cell therapy could restore blood and immune functions and reinstate immune tolerance. 

Pavel-Dinu is now an assistant professor in the Department of Pediatrics, Division of Immunology, at the University of Washington School of Medicine. In addition, she is a Principal Investigator at the Center for Immunity and Immunotherapies (CIIT), Seattle Children’s Research Institute (SCRI). 

“The concept behind the therapeutic application of clustered regularly interspaced short palindromic repeats [CRISPR]-based genome editing technology lays the foundation of my studies. We can now modify faulty DNA sequences in hematopoietic stem cells (tissue-specific stem cells) to restore the function of a gene necessary for normal immune or blood functions," she said. "The CRISPR/Cas9 nuclease is designed to recognize and mark by introducing a DNA break at the specific location in the genome where the DNA sequence is altered. This event triggers the cells to repair the break. Through the course of repair, we introduce the desired corrected DNA sequence through a non-pathogenic adeno-associated virus of serotype 6 (AAV6). My laboratory is interested in understanding how this process influences stem cells' regenerative functions."

Pavel-Dinu is now studying how oxygen rewires stem cells’ metabolism during culture and in the process of ex vivo CRISPR/Cas9-AAV6 gene correction. “In my newly established laboratory at the Center for Immunity and Immunotherapies at Seattle Children’s Research Institute, my team and I are studying the impact of ambient oxygen [21% O2], which is a non-physiological oxygen that hematopoietic stem cells are exposed to during the culture time,” she said. “These studies are a continuation of the work funded by the AABB Foundation award.” 

She noted that HSCs live in low oxygen inside the bone marrow niche. Yet, cells are cultured in higher oxygen concentrations in the laboratory. This difference in oxygen levels changes the metabolic makeup of the cells — and impacts the cells’ therapeutic functions. “For example, we observed that culturing HSCs in low oxygen slows the cells down, transcriptionally and metabolically, without impeding the effectiveness of the genome editing process,” she said. However, oxygen alone is only part of the story.  

“We also investigate the nutrient requirements during the culture and in response to stresses imposed by the CRISPR system,” Pavel-Dinu added. “The physical structure of the bone marrow has long been known for its critical role in the development and function of stem cells. We are working to recapitulate and study the bone marrow microenvironment in our laboratory and establish a unique and optimal culture setting to heal the defective HSCs.”   

There may be other factors that affect the longevity of HSCs. Pavel-Dinu said that her team is exploring how signaling pathways that sense metabolic stresses and regulate the cellular cycle work together to maintain the regenerative potential of HSCs. These networks could be tuned up or down by controlling the oxygen levels. These responses can rewire HSCs' cellular metabolism influencing HSCs’ identity, function, and ultimately their therapeutic potential. 

Pavel-Dinu's early-career grant from the AABB Foundation has been key to moving her research forward. The award “has helped me generate the necessary preliminary data to compete for larger funding awards from NIH,” she said. 

Moving forward, Pavel-Dinu plans to determine the full extent to which exposure to extreme oxygen levels alters cellular metabolism, the transcriptome, and the epigenome of hematopoietic stem cells.  

“We hope this knowledge will give us a deeper insight into how the process of regeneration is imprinted in these rare hematopoietic stem cells, and how it is lost in diseases like bone marrow failure, anemia and immune dysregulations or to activate as is in cancers of blood and immune origins,” she added. 

Pavel-Dinu noted that this fundamental research in hematopoietic regeneration has the potential to advance stem cell transplantation to benefit a broader range of blood and immune diseases and a wider patient population.  “We don’t just want to re-write the DNA sequence in patients with inherited defects but also restore stem cell's function caused by bone marrow microenvironment defects, as is in the case of auto-inflammatory diseases,” she said. 

However, it’s still in the early days and clinical use is years away.  

“We aim to complete and publish our studies in the next three to five years,” said Pavel-Dinu. “This work will form the basis for our IND-enabling research to advance ex vivo CRISPR/Cas9 with AAV6-based gene therapy to the clinic for RAG2 deficiency. It is a fatal disease if not treated in the first year of life, and it is caused by defects in our genome editing machinery necessary to establish immune function and tolerance.”