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Mutations in the gene encoding perforin are associated with familial HLH, called familial HLH type 2 (FHL2). Very little is known about how perforin works and the pathologic consequences of disease-associated mutations. We utilize murine and human cell culture models to study folding, trafficking, secretion and lipid binding of wild-type and mutant perforins.


HLH (of all genetic etiologies) has a 20-50% mortality rate despite aggressive immunosuppressive therapy and bone marrow transplantation (BMT); therefore, we are eager to identify additional therapeutic interventions that improve cytotoxic function in patients with HLH. Gene transfer (also known as gene therapy) offers the capacity to restore cytotoxicity and therefore eliminate antigen-presenting cells (APCs) by restoring the abnormal proteins of the cytotoxic pathway. Currently, none of our therapeutic maneuvers target this first step in the inflammatory cascade. Instead, current treatments focus on suppressing immune cells after persistent activation by APCs. Our long-term goal is to develop a gene therapy program for patients with all forms of genetically defined HLH. In the current project, we will test the feasibility of gene transfer to correct perforin deficiency in patient-derived cytotoxic lymphocytes.

Dr. Punam Malik, Dr. Filipovich, and Dr. Michael Jordan.

Dr. Malik is the Program Leader for the Molecular and Gene Therapy Program at Cincinnati Children’s. She is currently coordinating a clinical trial for gene therapy in beta thalassemia and sickle cell anemia. She directs the viral vector production lab that is the exclusive producer of clinical grade self-inactivating (SIN) lentivirus driven by a cellular promoter for the international trial for X-linked severe combined immunodeficiency (SCID), already underway.

Dr. Filipovich is the clinical director of the Immunodeficiency and Histiocytosis Program at Cincinnati Children’s and also a co-investigator on the X-linked SCID gene therapy trial. She and Dr. Jordan will assist in identifying and consenting patients with FHL2 so that we may obtain blood and bone marrow samples for research.

In addition, Dr. Jordan is testing the utility of the lentiviral vectors in a pre-clinical murine model of FHL2.


Many of the gene defects identified in patients with FHL2 are missense mutations that render the perforin protein misfolded or unstable. There are reports in the literature that hypomorphic perforin missense mutants can be rescued following incubation of cells at lower temperatures. Our lab also observed that glycerol can restore the expression and functions of certain perforin mutants (unpublished data). These observations support the possibility that some available drugs may be able to restore the expression or function of hypomorphic mutant perforins in patients with HLH. Remarkably, small-molecule compounds that rescue the function of CFTR (cystic fibrosis transmembrane conductance regulator) mutants in cystic fibrosis (CF) patients have been successfully identified by high throughput screening, and one of those, the Ivacaftor (VX-770), has been approved recently by FDA to treat CF patients with the G551D mutation. CFTR mutations also lead to protein misfolding or mistrafficking, which is very similar to perforin mutations that cause HLH.

Our current project utilizes a novel NK cell cytotoxic assay, (i.e. luminescence-based granzyme B biosensor assay) in a high throughput approach to screen and identify drugs that can enhance cytotoxic function of NK cells from HLH patients. High throughput screening is an approach widely used in new drug development. However, high throughput screening of NK cell function has been hindered by lack of a suitable assay. The conventional gold standard for NK cell cytotoxicity, Cr51 release assay, cannot be used in this context because of the radioactive isotope. We have developed a luminescence-based granzyme B biosensor to measure NK cell cytotoxicity and have executed the first robotic screen of a small compound library. The hits from this screen await confirmation testing in a Cr51 assay, and we aim to execute a broader screen and identify mechanisms for enhancement.

NIH Molecular Libraries Small Molecule Repository


We hypothesize that individual variation in lymphocyte cytotoxicity is influenced by both immunologic triggers (pathogens, vaccines, haptens) and genetic variation in proteins that positively and negatively regulate the cytotoxic response. We predict that proteins that “fine tune” cytotoxic function may be identified by unbiased shRNA knockdown of all cDNA in CTLs using a high throughput screening approach. This knockdown may be accomplished by expression of the entire murine shRNA library in CTLs and screening for cytotoxicity. Using this approach, we expect to identify novel proteins that are 1) critical for killing (i.e. cause decreased cytotoxic function with knockdown) or 2) actively limiting the cytotoxic response (i.e. cause enhanced killing with knockdown). As only half of the patients with HLH and related disorders have an identifiable genetic diagnosis after screening for known mutations, there is certainly a need to identify new genes that may be disease causing or disease modifying. Therefore, the results of screening may enhance diagnosis in patients with HLH, chronic viral infections, and related lymphoproliferative disorders.