Hemant Khanna, Ph.D.
Research Projects

Ciliopathies are a group of developmental and progressive disorders caused by defects in proteins involved in regulating primary cilia formation or function. The Khanna lab employs a multi-pronged approach to studying cilia-dependent pathways and their association with retinal degeneration in X-linked retinitis pigmentosa (XLRP), a clinically and genetically heterogeneous ciliary disorder characterized by severe loss of vision at an early age. Because a majority of retinal degenerative diseases are due to defective outer segment disc shedding and renewal, our lab also works on understanding the extrinsic and intrinsic cues that regulate gene expression in photoreceptors during outer segment disc morphogenesis. A major focus is also on identifying potential modifier genes that affect the penetrance and severity of the retinal phenotype in syndromic ciliopathy patients and on understanding the biochemical mechanism of the disease.

Our approach includes:

1. Genetic studies
   Although 6 loci for XLRP have been mapped to date, only two genes, Retinitis Pigmentosa GTPase Regulator (RPGR) and RP2 have been cloned.  We are identifying additional XLRP-associated genes by further refining the disease loci and by analyzing candidate genes.  Our studies also include identification of genes associated with X-linked cone-rod degeneration families.
   
   
2. Interactome analysis
   To gain mechanistic insights into RPGR and RP2 function, we are identifying RPGR- and RP2-interacting proteins in photoreceptor cilia by yeast two-hybrid and co-immunoprecipitation & Mass Spectrometry analyses.  We have identified a number of potential RPGR- and RP2-interacting proteins, which are either mutated in Leber congenital amaurosis (a childhood blindness disorder), Joubert Syndrome and Meckel-Gruber Syndrome or are excellent candidate ciliopathy genes.  
   

   Localization of a novel RPGR-interacting protein (red) to photoreceptor inner sebment (IS) and connecting cilium (CC), as co-stained with acetylated alpha-tubulin (green). Nuclei are stained with DAPI (blue). OS: outer segment

   
   
3. Animal models
   To better understand the pathogenesis of XLRP and other X-linked retinal diseases, we are generating mouse and zebrafish models representing the human phenotype.  Our analysis includes assessment of age-dependent photoreceptor degeneration and examination of defects in protein trafficking in photoreceptors.  In vivo gene knockdown mouse models are also being generated using Cre/loxp system or by subretinal injection of shRNA -encoding viral vectors.

   Histological section of retina from zebrafish embryo. Wild type retinas at 3dpf have fully laminated retinas with distinct ganglion cell layer (GCL), inner nulcear layer (INL) and outer nuclear layer (ONL).

   
   
4. In vitro models
   To assess the biochemical function of RPGR and RP2, we are generating in vitro models of Rpgr- and Rp2- knockdown by using shRNA technology in neuronal as well as non-neuronal cell lines.  We are also performing reciprocal experiments to knockdown the interacting partners to assess the effect on the localization and function of RPGR and RP2.  We are also examining cilia-mediated signaling and regulation of intrinsic gene expression in response to extrinsic stimuli to better understand the signaling events that occur in response to photoreceptor disc shedding and renewal.
   
   

   hTERT RPE-1 cells stained for RPGR (red), acetylated a-tubulin (green; a ciliary marker), and DAPI (blue)

   
   
5. Therapeutic strategies
   In collaboration with clinician scientists, we are developing effective therapeutic modalities to treat photoreceptor degenerative diseases.  We are employing cell-based transplantation strategies to replace dying photoreceptors with precursor cells that have the ability to integrate and differentiate into mature photoreceptors.  Mouse models of early- as well as late-onset retinal degeneration are being employed for these studies.