Pavel-Dinu Laboratory

Leading Research for Gene and Cell Therapies for Patients with Inborn Errors of Immunity

The Science

In Pavel-Dinu Lab, we combine regenerative biology with genome editing technology to rewrite the DNA code of a defective gene and restore its function. Our genome editing platform can effectively and precisely modify the DNA sequence, at nucleotide resolution, in hematopoietic stem cells (HSCs) cultured outside the body (ex vivo) to restore the immune development and function following transplantation (in vivo). This targeted therapeutic approach has revolutionized medicine for rare blood diseases (e.g., sickle cell disease). However, patients with rare immune diseases have yet to benefit from it. This is where our work begins.

Rare Diseases: There are 6,000 – 10,000 unique rare diseases worldwide. In United States alone, an estimated 30 million people suffer from one form of rare disease. In other parts of the world the prevalence of rare diseases is even higher, with 1-5 per 10,000 life births. Rare diseases are a significant burden on human health, and to the healthcare industry with nearly $400 billion being spend annually in total direct medical costs.

72% of rare diseases are due to defects in a single gene (mono-genetic) and affect a broad range of systems and organs.

69% of rare mono-genetic diseases have a pediatric onset with 3 out of 10 children die before their 5th birthday.

95% of rare mono-genetic immune diseases collectively referred to as Inborn Errors of Immunity (IEI) do not have an FDA approved treatment.

The lack of treatment options that safely, effectively, and permanently restore immunity in patients underscores the need to continue our research efforts to address this unmet medical need.

Rare Disease Day observed on February 28

Our research mission is to discover the complex processes of hematopoietic stem cell regeneration, understand how it goes awry in IEIs, and turn our scientific insights into innovative and long-lasting gene and cell therapies accessible by an ethnically and age-diverse community of patients.

RESEARCH PROJECTS

When stem cells need a tune-up

The study of human hematopoietic regeneration is hampered by the limited number of HSCs in our bodies, the inability to expand these rare cells outside the body, and, for ethical reasons, using patients’ cells for research purposes only. IEIs are a growing class of rare mono-genetic immune diseases that add great value to our scientific endeavors and have vast implications for advancing medicine. From the root cause to the molecular mechanism and disease progression, IEIs offer the complete disease layout for us to study the complex biological processes of regeneration, uncover new principles, and transform how we approach medicine in the 21st century.

Our laboratory is built on a solid technical and conceptual framework for applying genome editing technology to modify DNA sequences in hematopoietic stem cells and restore gene function. When working in the group of Matthew Porteus at Stanford University, we have shown that genome editing can precisely correct any pathogenic mutation in the interleukin 2 receptor gamma (IL2RG) gene causing X-linked severe combined immunodeficiency (SCID-X1), also known as the “Bubble Boy” disease (Nature Communications). Our study further demonstrated that up to 20% of genome-editing hematopoietic stem cells survived for 5 months in mouse bone marrow, the gold standard for evaluating long-term human hematopoietic regeneration.

This study holds significant implications for the gene therapy field: it demonstrated that long-term HSCs (multipotent cells) are amenable to genome editing. However, only a limited number of cells survive the ex vivo genome editing procedure to support in vivo hematopoiesis. Depending on the disease indication, this loss in regenerative potential may not be sufficient to sustain patient life-long therapy for all disease indications. We work to make genome editing technology compatible with hematopoietic regeneration to advance its therapeutic application to a broader range of diseases.

The genome editing gene correction of hematopoietic stem cells approach becomes a critical consideration when developing novel therapies for immune diseases that carry a risk of genomic instability if the gene’s endogenous regulation is not preserved. This is the case for the Recombination Activating Gene 2 (RAG2), a protein with endonuclease (an enzyme that cuts DNA) activity (Journal of Clinical Immunology). RAG1 and RAG2 are part of a larger protein complex essential during the development and function of lymphoid cells (T and B-cells). These proteins generate a diverse (polyclonal) receptor repertoire through a “cut” and “paste” approach that shuffles DNA sequences to create unique receptors’ epitopes capable of detecting and mounting an immune response against a broad range of antigens. The RAG1/2 protein complex, in a sense, functions as the genome editing machinery for the adaptive immune system.

Infants born with RAG1 or RAG2 deficiency develop life-threatening immune diseases ranging from SCID to severe forms of autoimmune and inflammatory conditions. While part of Matthew Porteus research team we showed that targeted correction of hematopoietic stem cells carrying a null mutation (0% endonuclease activity) in the RAG2 gene corrects the SCID phenotype by restoring the endonuclease function, generating diverse receptors on the developing T and B lymphocytes and restoring the NK cells’ developmental block (Blood Advances). In our lab, we focus on Omenn Syndrome caused by hypomorphic mutations in the RAG2 gene (5% – 30% endonuclease activity compared to normal gene) resulting in life-threatening forms of autoimmune and inflammatory conditions. Infants born with RAG2-OS require hematopoietic stem cell transplantation in the first year of life. We study the broader hematopoietic defects in RAG2-OS diseases and evaluates the extent an autologous ex vivo genome editing therapy can offer a permanent and safe therapy.

By studying rare mutations in rare mono-genetic immune diseases, our laboratory deconstructs the networks and mechanisms that regulate hematopoietic regeneration in healthy and how anomalies in hematopoietic regeneration drive cellular and tissue dysfunction in immune diseases.

Keeping stem cells resilient

Hematopoietic stem cells reside inside the bone marrow. From birth to teens, stem cells frequently regenerate to establish hematopoiesis. As we enter adulthood and age, the process of hematopoietic regeneration slows down and is less efficient at producing immune cells. This observation holds important implications for cell replacement therapies where adult hematopoietic stem cells are used for allogeneic (not-self) or autologous (self) transplantations, such as during ex vivo genome editing-based gene therapies.

In the current state, ex vivo genome editing is a stressful and damaging procedure for hematopoietic stem cells. Cells are removed from their bone marrow niche microenvironment and exposed to non-physiological conditions. Ex vivo culturing modalities do not recapitulate the cellular, metabolic, and structural environment of the human bone marrow that hematopoietic stem cells are used to.

A tightly framed view of bone marrow tissue with visible cell nuclei, intricate structure of hematopoietic cells

While significant advancement has been made to deliver genome editing tools to the cells, our understanding of how the stem cells respond to genome editing manipulations and adapt to ex vivo non-physiological conditions is largely unknown. Genome editing is an energetically demanding process for hematopoietic stem cells. Stem cells are under increased proliferative demands, a process that is tightly linked to cells’ ability to acquire nutrients, generate metabolic energy (ATP), and drive anabolism, particularly for nucleotide and nucleic acid biosynthesis necessary during nuclear DNA repair and mitochondria DNA synthesis. This may lead to exhaustion, excessive differentiation, or premature cell death.

Metabolism has a recognized role in hematopoietic regeneration and immune development and function. A disrupted metabolism in hematopoietic stem cells may thus lie at the root of many degenerative, age-related immune diseases (Blood) and one of the culprits driving loss of hematopoietic regeneration during ex vivo culture and genome editing. Uncovering hematopoietic stem cells’ metabolic needs and vulnerabilities to advance science and therapies sets the stage for our current studies.

How rare immune cells keep stem cells fit

Like hematopoietic stem cells, invariant Natural Killer T (iNKT) cells are a rare cell population. iNKT cells have features of innate and adaptive immune cells and hold broad immunomodulatory properties. They orchestrate immune homeostasis and the adaptive and innate immune responses to pathogens through interleukins, growth factors, and chemokines. Their regulatory role extends to almost all hematopoietic cells.

It is an emerging paradigm that iNKT cells regulate early hematopoiesis, by providing (direct and indirect) cues that stimulate self-renewal and mediate differentiation. Reduced or absent iNKT cells drive susceptibility to infection, early-onset inflammation, and organ-specific autoimmunity as part of the disease pathology in some rare mono-genetic immune diseases (e.g., DOCK8 deficiency, WAS, XLP disease). Our laboratory is focused on investigating the HSCs’ fitness in an iNKT deficiency background to advance our understanding of how immune cells may regulate hematopoietic regeneration and guide the optimal therapeutic strategy for patients lacking iNKT cells.

Adaptive and Innate immunity. Cells of The Immune System.

Over 20 clinical trials are evaluating iNKT-based immunotherapies for the potential to restore hematopoiesis disrupted by excessive self-renewal leading to cancer. No trials to date are testing adoptive iNKT cell transfer to treat autoimmune and inflammatory conditions. Our research interests address fundamental questions in iNKT biology in healthy and rare mono-genetic immune diseases (IEIs) to study (i) how iNKTs regulate self-renewal of hematopoietic stem cells, (ii) to advance adoptive iNKT cell therapy to treat autoimmune and inflammatory diseases (rare and common) and (iii) to restore and maintain immune tolerance following stem cell transplantation.

Healing the immune system

Understanding the mechanisms and networks that disrupt hematopoietic regeneration is crucial for developing effective treatments for immune perturbations. Our lab is dedicated to this pursuit, focusing on the potential of genome editing technology as a reliable therapeutic strategy to correct rare genetic defects in hematopoietic stem cells and infuse the corrected cells back in the patient to restore immunity. In collaboration with the group of David Rawlings, the director of the Center for Immunity and Immunotherapies (CIIT), Matthew Porteus, the director of the Center for Definitive and Curative Medicine (CDCM) at Stanford University, Luigi Notarangelo, Chief of the Laboratory of Clinical Immunology and Microbiology (NIH/NIAID), and Anna Villa, San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), we aim to develop an FDA-approved autologous ex vivo genome editing cell therapy for patients with RAG2 deficiency.

Medical capsule and transparent cell, 3d rendering.

Our laboratory is also working to develop an adoptive cell therapy (ACT) using off-the-shelf iNKT cells with tolerogenic properties to restore immune tolerance. This immunotherapeutic approach relies on purifying and enriching subsets of iNKT cells that enhance TH2 immunity or T regulatory (Treg) functions or suppress TH1 and TH17 responses. Our research efforts are centered on developing a cell-based therapy to benefit patients with acute graft-versus-host-disease (aGvHD) or as the first line of treatment for patients with active inflammatory and autoimmune diseases refractory to other therapies.

We harness hematopoietic regeneration and unlock the potential of immune cells to bring life-changing medicine to improve human health.