- Repair of Corneal Injury with Stem Cell-Based Bioengineered Tissue
- Targeted Therapy for Treating Polycystic Kidney Disease
- Development of Augmented Leukemia/Lymphoma-Specific T-Cell Immunotherapy for Deployment with Haploidentical Hematopoietic Progenitor-Cell Transplant
- Pharmacological Evaluation of the Novel Analgesics
Among the many dangers to soldiers on the battlefield is the risk for eye injury or infection, as explosions and other threats can cause mechanical, thermal, chemical, microbial, and radiation damage to the cornea that can lead to impaired vision or blindness. The cornea serves as the primary defense for the eye and is maintained by limbal stem cells that may be cultivated for transplantation to heal the damaged eye. Approximately 110 military patients per year seek treatment for a damaged cornea, but there is a shortage in donor corneal tissue for this regenerating procedure. Dr. De Quan Li of the Baylor College of Medicine received a Fiscal Year 2006 Investigator-Initiated Research Award from the Peer Reviewed Medical Research Program to engineer stem cell-based corneal constructs using human limbal epithelial progenitor cells (LEPC) to improve availability of this vision saving transplantation procedure. Dr. Li optimized the procedure for isolating human LEPC from limbal epithelial tissues or their primary cultures and selected cells for corneal tissue engineering based on a relatively undifferentiated state, high proliferative potential, and regenerative capacity. He then optimized a cell culture system for expanding the LEPC ex vivo, using a customized culture medium and a feeder cell layer composed of human fibroblast cell lines. To complete the construct, Dr. Li fabricated lamellar corneal stromal discs of optimal size (10-11 mm diameter) and thickness (100 µm) to cover the entire surface of the recipient cornea. These discs serve as the stromal substrate for the LEPC, facilitating the regeneration of a normal corneal epithelium. Biomarker analyses verified that this artificial epithelium has a very similar phenotype to naturally occurring corneal epithelium, suggesting that it may also function like the native cornea. Additional experiments demonstrated that the LEPC corneal constructs maintain regenerative capacity, completely healing alkaline burn wounds within 2-4 days in culture. Together, these results indicate that bioengineered corneal constructs may significantly impact the clinical treatment of eye injuries, with the potential to regenerate damaged corneas and ultimately restore vision.
Figure 1. Phenotype of artificial corneal epithelium generated on donor stromal disc. Representative images of immunofluorescent staining for corneal epithelial markers (Cx43, K3, involucrin, ABCG2, p63, integrin ß1 and EGFR) with Hematoxylin-eosin (HE) and Hoechst 33342 nuclear counterstaining on frozen sections of the artificial corneal construct (A), in comparison with those from donor tissues, limbus (L) and cornea (C).
Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in either the PKD1 or PKD2 gene, is the most common life-threatening hereditary disease, with an incidence of 1/400-1/1000. ADPKD leads to renal epithelial cyst formation, fibrosis, and progressive destruction of kidney function. With no treatment available, most ADPKD patients eventually require life-long dialysis or kidney transplantation. Dr. Thomas Weimbs' research team has previously shown that PC1, the product of the PKD1 gene, interacts with mammalian target of rapamycin (mTOR) and tuberin, a negative regulator of mTOR. Also, they found that mTOR activity is highly upregulated in renal cysts in ADPK compared to surrounding normal cells, and treating animal models with the mTOR inhibitor rapamycin was successful at specifically treating the cysts without affecting the adjacent normal tubule cells. Unfortunately, rapamycin was unsuccessful in human clinical trials when using the lower, more tolerable doses necessary to avoid immunosuppression. Frustrated by the inability to treat ADPKD with tolerable doses of rapamycin, Dr. Weimbs designed a conjugated form of rapamycin that could be targeted to the folate receptor on the kidney cells and avoid systemic effects.
With funding from a Fiscal Year 2006 Peer Reviewed Medical Research Program Investigator-Initiated Research Award, Dr. Weimbs tested this conjugated form of rapamycin (EC0371/FC-rapa), which is taken up via folate receptor-mediated endocytosis and then cleaved intracellulary to reconstitute the active drug. His research showed that folate receptor is highly expressed in renal cyst-lining cells in human ADPKD tissue and two mouse PKD models, and in vitro testing showed that FC-rapa inhibits mTOR activity in a dose- and folate receptor-dependent manner. Additionally, treatment of PKD mouse models with FC-rapa resulted in inhibition of mTOR activity, inhibition of renal cyst growth, and preservation of renal function. Renal cyst growth was even inhibited in a 10X lower dose of FC-rapa in mouse models, providing kidney-specific inhibition of the mTOR pathway without causing detrimental systemic effects. Dr. Weimbs' findings suggest that FC-rapa has the potential to alleviate renal cystic disease in humans without the complications of systemic immunosuppression, providing hope that the first treatment for ADPKD is on the horizon. Moreover, this study suggests that, in addition to ADPKD, the targeting of folate-conjugated compounds to kidneys may be a promising approach for treating other renal epithelial disorders.
Olsan EE, Mukherjee S, Wulkersdorfer B, Shillingford JM, Giovannone AJ, Todorov G, Song X, Pei Y, and Weimbs T. 2011. Signal transducer and activator of transcription-6 (STAT6) inhibition suppresses renal cyst growth in polycystic kidney disease. Proceedings of the National Academy of Sciences of the U S A 108:18067-18072.
Weimbs T. 2011. Third-hit signaling in renal cyst formation. Journal of the American Society of Nephrology 22(5):793-795.
Torres VE, Boletta A, Chapman A, Gattone V, Pei Y, Quian Q, Wallace DP, Weimbs T, and Wüthrich RP. 2010. Prospects for mTOR inhibitor use in patients with polycystic kidney disease and hamartomatous diseases. Clinical Journal of the American Society of Nephrology 5(7):1312-1329.
Shillingford JM, Piontek KB, Germino GG, and Weimbs T. 2010. Rapamycin ameliorates PKD resulting from conditional inactivation of Pkd1. Journal of the American Society of Nephrology 21:489-497.
Development of Augmented Leukemia/Lymphoma-Specific T-Cell Immunotherapy for Deployment with Haploidentical Hematopoietic Progenitor-Cell Transplant
Posted May 8, 2012
Laurence Cooper, Ph.D. University of Texas MD Anderson Cancer Center
Although allogeneic hematopoietic progenitor-cell transplant (HPCT) may eradicate high-risk B-cell malignancies, including lymphoma and leukemia in pediatric patients, HLA (human leukocyte antigen)-matched siblings or unrelated donors are not readily available for every child. Due to the limited availability of HLA-identical donors, pediatric patients may benefit from transplantation of haploidentical (or half-matched) HPCs that are more widely accessible. While this procedure is potentially lifesaving, pediatric haploidentical recipients remain at significant risk for disease relapse and opportunistic infection. Recent clinical studies have demonstrated, however, that infusion with donor-derived T cells genetically modified to target antigens expressed by opportunistic pathogens can protect against infection after allogeneic HPCT. Based on these findings, Dr. Laurence Cooper hypothesized that treatment with donor-derived T cells genetically modified to target the CD19 antigen expressed by malignant B cells may improve the relapse-free survival rate of pediatric haploidentical HPCT recipients with leukemia or lymphoma. Therefore, with funding from a fiscal year 2006 Advance Technology: Product/Technology Down-Selection or Optimization Award, Dr. Cooper's research team developed novel methodology to generate CD4+ and CD8+ T cells that can eradicate malignant CD19-expressing B cells in a mouse model. Although infusion with HLA half-matched T cells can result in the development of a serious complication called graft-versus-host disease, Dr. Cooper demonstrated that blockage of CD28 prior to T-cell treatment limits the development of this life-threatening complication. Dr. Cooper has utilized these exciting preclinical results to obtain other funding with which he is currently evaluating this technology in a first-in-human clinical trial.
Pain management has been a major focal point of both the civilian and military medical communities over the past 10 years. To date, opiate analgesics that act through the mu opioid receptor, such as morphine, remain the most effective approach to managing moderate to severe pain. Unfortunately, these drugs also have considerable side-effects including respiratory depression, nausea, vomiting, itching, and constipation. More importantly, opiates have a high propensity for abuse, with tolerance and physiological dependence developing rapidly, thus making them impractical for the long-term treatment of chronic pain. Alternative pain medications that act via kappa and delta opioid receptors are neither as effective as morphine-like opiates, nor are they devoid of secondary complications, as they are known to have dysphoric effects. The onrush of injured Service members returning from the conflicts abroad highlighted the urgency to develop more effective means to treat pain in both its acute and chronic forms. As such, identification of novel painkillers with reduced potential for abuse and fewer side-effects is of critical importance.
The nociceptin (NOP) receptor, also known as the orphanin FQ receptor, is the fourth member of the opioid receptor family. Although, as the name implies, the NOP receptor is known to play a role in regulating the biological response to noxious stimuli, its effectiveness as a potential treatment target for moderate to severe pain management is still to be determined. Nociceptin/orphanin FQ (N/OFQ), the endogenous peptide selective for the NOP receptor, has been shown to have actions similar to other opioid peptides at the cellular level. However, studies in mice have yielded disparate results, revealing that N/OFQ function as an analgesic can be either similar or opposite to that of other opiates depending on the location of the receptor within the central nervous system. Dr. Mei-Chuan Ko received a Fiscal Year 2006 Peer Reviewed Medical Research Program (PRMRP) Investigator-Initiated Research Award to investigate, in other animal models, whether NOP receptors can serve as potential analgesic targets without the severe side-effects observed with other opiates. He found that spinal administration of NOP receptor agonists produced antinociceptive effects comparable to those of other clinically used mu receptor opiates without eliciting secondary side-effects like itching or sedation in three different primate pain models. Furthermore, the withdrawal symptoms produced after cessation of chronicly administered NOP receptor agonists were significantly less severe than those observed with morphine. An additional important finding from this study was that the NOP receptor agonist Ro 64-6198 did not cause respiratory depression at doses larger than the antinociceptive dose. Altogether, these results suggest that the NOP receptor may be a promising target for the treatment of acute and chronic pain without the dangerous side effects commonly associated with opiate pharmacology.
Ko MC and Naughton NN. 2009. Antinociceptive effects of nociception/orphanin FQ administered intrathecally in monkeys. Journal of Pain 10:509-516.
Ko MC, Woods JH, Fantegrossi WE, Galuska CM, Wichmann J, and Prinssen EP. 2009. Behavioral effects of a synthetic agonist selective for nociception/orphanin FQ peptide receptors in monkeys. Neuropsychopharmacology 34:2088-2096.
Hu E, Calo G, Guerrini R, and Ko MC. 2010. Long-lasting antinociceptive spinal effects in primates of the novel nociception/orphanin FQ receptor agonist UFP-112. Pain 148:107-113.
Podlesnik CA, Ko MC, Winger G, Wichmann J, Prinssen EP, and Woods JH. 2011. The effects of nociception/orphanin FQ agonist Ro 64-6198 and diazepam on antinociception, remifentanil self-administration, and anxiolytic-like responding in rhesus monkeys. Psychopharmacology 213:53-60.