Paul J. Park

The Sheep Model of in utero Gene Therapy

Once its full clinical potential has been realized, hematopoietic stem cell based gene therapy (GT) promises to cure a wide array of both inborn and acquired diseases. For many genetic disorders, early onset and irreparable tissue and organ damage necessitate innovative methods that allow therapeutic intervention early in development, if a full cure is to be realized. Performing GT in utero would allow early correction prior to disease onset and is thus one of the few therapeutic modalities that could promise the birth of a healthy infant. Several features of the developing fetus may circumvent obstacles that have thus far been observed in GT trials. For example, the immune naïveté of the early gestational fetus may evade immune reactions to the vector and transgene product. Furthermore, fetal exposure to foreign antigens can result in sustained tolerance, suggesting that induction of tolerance to the vector/transgene product could allow postnatal treatment to be performed successfully. In addition to these immunologic advantages, the fetal hematopoietic system promises to be more amenable to retrovirus-mediated gene transfer than either the neonate or adult as a result of both proliferation and expansion of the stem/progenitor cell pool that take place during fetal development. To investigate whether these characteristics of the developing fetus could be used to advantage to efficiently transduce hematopoietic stem cells, we developed an approach to in utero GT, in which retroviral vectors were directly injected into the peritoneal cavity of preimmune fetal sheep. This approach resulted in the transfer and long-term (>5 years) expression of exogenous genes within the hematopoietic system of primary and secondary recipients, albeit at relatively low levels that would not likely be therapeutic in most diseases. These studies also demonstrated that the direct injection of retroviral vectors into preimmune fetal sheep results not only in the successful transduction of long-term engrafting hematopoietic stem cells, but also in the widespread distribution of vector to all other tissues examined, including the reproductive organs. In an effort to increase the hematopoietic cell transduction to clinically relevant levels, we repeated our initial studies with 1,000-fold higher titer vectors. This led to only a modest (two- to fourfold) increase in the transduction levels, suggesting that factors other than absolute vector dosage were responsible for the low levels of gene transfer. For this reason, we have more recently begun evaluating the effect of recipient gestational age on the efficiency of gene transfer to both hematopoietic and nonhematopoietic tissues. Thus far, we have observed an inverse relation between the gestational age at the time of vector administration and the level of transduction and expression of the transgene within the hematopoietic system, such that fetuses injected earlier in gestation have higher levels of hematopoietic cell transduction. These elevated levels have persisted for at least 1 year after injection, suggesting that the enhancement is at the level of primitive stem/progenitor cells. When analyzing the liver sections from animals that had received the vector at different gestational ages, we also observed an inverse correlation between recipient age and efficiency of gene transfer to the hepatocytes, such that a high efficiency of gene transfer occurred at early ages, while very little occurred at later stages of gestation. In contrast to the findings in the hematopoietic system and in the liver, analysis of the lungs of these same animals revealed that the efficiency of transduction of nonhematopoietic lung tissue increased with increasing gestational age. These results demonstrate that both hematopoietic cells and nonhematopoietic cells within liver and lung are transduced following direct injection of murine retroviral vector supernatants into the peritoneal cavity of preimmune fetal sheep and suggest that the developmental stage of each organ at the time of injection may determine its susceptibility to in utero gene transfer.

Radiosurgery for large-volume (> 10 cm3) benign meningiomas

OBJECT Stereotactic radiosurgery (SRS) has proven to be a safe and effective treatment for many patients with intracranial meningiomas. Nevertheless, the morbidity associated with radiosurgery of larger meningiomas is poorly understood. METHODS The authors performed a retrospective review of 116 patients who underwent SRS for meningiomas (WHO Grade I) > 10 cm3 between 1990 and 2007, with a minimum follow-up of 12 months. Patients with atypical or malignant meningiomas and those who received prior radiotherapy were excluded. The average tumor volume was 17.5 cm3 (range 10.1-48.6 cm3); the average tumor margin dose was 15.1 Gy (range 12-18 Gy); and the mean follow-up duration was 70.1 months (range 12-199 months). RESULTS Tumor control was 99% at 3 years and 92% at 7 years after radiosurgery. Thirty complications after radiosurgery were noted in 27 patients (23%), including 7 cases of seizures, 6 cases of hemiparesis, 5 cases of trigeminal injury, 4 cases of headaches, 3 cases of diplopia, 2 cases each of cerebral infarction and ataxia, and 1 case of hearing loss. Patients with supratentorial tumors experienced a higher complication rate compared with patients with skull base tumors (44% compared with 18%) (hazard ratio 2.9, 95% CI 1.3-6.7, p = 0.01). CONCLUSIONS The morbidity associated with SRS for patients with benign meningiomas > 10 cm(3) is greater for supratentorial tumors compared with skull base tumors. Whereas radiosurgery is relatively safe for patients with large-volume skull base meningiomas, resection should remain the primary disease management for the majority of patients with large-volume supratentorial meningiomas.

Early fetal gene delivery utilizes both central and peripheral mechanisms of tolerance induction.

OBJECTIVE We previously reported the induction of stable immune tolerance following direct injection of retroviral vectors into preimmune fetal sheep. In the present studies, we conduct detailed analysis of the thymus of recipients of in utero gene transfer (IUGT) to delineate the mechanism of the observed immune tolerance and assess the impact of recipient age on this process. MATERIALS AND METHODS Fetal sheep at varying gestational ages received the MSCV-NeoR-RFP retroviral vector. The thymus was then collected from these animals at 27 to 30 days postinjection and analyzed for evidence of transduction of key immunoregulatory thymic cells. RESULTS Our results reveal that both thymic epithelial cells (TEC), crucial for presentation of self-antigen during T-cell thymic selection, and the cells comprising the Hassalls corpuscles, which can present antigen directly and also instruct dendritic cells to induce the formation of CD4(+)CD25(+) T-regulatory cells in the thymus, were only efficiently transduced if IUGT was performed early in gestation. CONCLUSIONS Our findings thus demonstrate, for the first time, that early IUGT can potentially take advantage of multiple tolerogenic avenues in the fetus, transducing both TEC, which promote central tolerance, and Hassalls corpuscles, which induce formation of T regulatory cells that could act to maintain peripheral tolerance to the transgene products.

Smooth Muscle Cell Genome Browser: Enabling the Identification of Novel Serum Response Factor Target Genes

Genome-scale expression data on the absolute numbers of gene isoforms offers essential clues in cellular functions and biological processes. Smooth muscle cells (SMCs) perform a unique contractile function through expression of specific genes controlled by serum response factor (SRF), a transcription factor that binds to DNA sites known as the CArG boxes. To identify SRF-regulated genes specifically expressed in SMCs, we isolated SMC populations from mouse small intestine and colon, obtained their transcriptomes, and constructed an interactive SMC genome and CArGome browser. To our knowledge, this is the first online resource that provides a comprehensive library of all genetic transcripts expressed in primary SMCs. The browser also serves as the first genome-wide map of SRF binding sites. The browser analysis revealed novel SMC-specific transcriptional variants and SRF target genes, which provided new and unique insights into the cellular and biological functions of the cells in gastrointestinal (GI) physiology. The SRF target genes in SMCs, which were discovered in silico, were confirmed by proteomic analysis of SMC-specific Srf knockout mice. Our genome browser offers a new perspective into the alternative expression of genes in the context of SRF binding sites in SMCs and provides a valuable reference for future functional studies.

Transcriptome of interstitial cells of Cajal reveals unique and selective gene signatures

Transcriptome-scale data can reveal essential clues into understanding the underlying molecular mechanisms behind specific cellular functions and biological processes. Transcriptomics is a continually growing field of research utilized in biomarker discovery. The transcriptomic profile of interstitial cells of Cajal (ICC), which serve as slow-wave electrical pacemakers for gastrointestinal (GI) smooth muscle, has yet to be uncovered. Using copGFP-labeled ICC mice and flow cytometry, we isolated ICC populations from the murine small intestine and colon and obtained their transcriptomes. In analyzing the transcriptome, we identified a unique set of ICC-restricted markers including transcription factors, epigenetic enzymes/regulators, growth factors, receptors, protein kinases/phosphatases, and ion channels/transporters. This analysis provides new and unique insights into the cellular and biological functions of ICC in GI physiology. Additionally, we constructed an interactive ICC genome browser (http://med.unr.edu/physio/transcriptome) based on the UCSC genome database. To our knowledge, this is the first online resource that provides a comprehensive library of all known genetic transcripts expressed in primary ICC. Our genome browser offers a new perspective into the alternative expression of genes in ICC and provides a valuable reference for future functional studies.

Loss of serum response factor induces microRNA-mediated apoptosis in intestinal smooth muscle cells

Serum response factor (SRF) is a transcription factor known to mediate phenotypic plasticity in smooth muscle cells (SMCs). Despite the critical role of this protein in mediating intestinal injury response, little is known about the mechanism through which SRF alters SMC behavior. Here, we provide compelling evidence for the involvement of SRF-dependent microRNAs (miRNAs) in the regulation of SMC apoptosis. We generated SMC-restricted Srf inducible knockout (KO) mice and observed both severe degeneration of SMCs and a significant decrease in the expression of apoptosis-associated miRNAs. The absence of these miRNAs was associated with overexpression of apoptotic proteins, and we observed a high level of SMC death and myopathy in the intestinal muscle layers. These data provide a compelling new model that implicates SMC degeneration via anti-apoptotic miRNA deficiency caused by lack of SRF in gastrointestinal motility disorders.

Serum Response Factor Is Essential for Prenatal Gastrointestinal Smooth Muscle Development and Maintenance of Differentiated Phenotype.

Background/Aims Smooth muscle cells (SMCs) characteristically express serum response factor (SRF), which regulates their development. The role of SRF in SMC plasticity in the pathophysiological conditions of gastrointestinal (GI) tract is less characterized. Methods We generated SMC-specific Srf knockout mice and characterized the prenatally lethal phenotype using ultrasound biomicroscopy and histological analysis. We used small bowel partial obstruction surgeries and primary cell culture using cell-specific enhanced green fluorescent protein (EGFP) mouse lines to study phenotypic and molecular changes of SMCs by immunofluorescence, Western blotting, and quantitative polymerase chain reaction. Finally we examined SRF change in human rectal prolapse tissue by immunofluorescence. Results Congenital SMC-specific Srf knockout mice died before birth and displayed severe GI and cardiac defects. Partial obstruction resulted in an overall increase in SRF protein expression. However, individual SMCs appeared to gradually lose SRF in the hypertrophic muscle. Cells expressing low levels of SRF also expressed low levels of platelet-derived growth factor receptor alpha (PDGFRαlow) and Ki67. SMCs grown in culture recaptured the phenotypic switch from differentiated SMCs to proliferative PDGFRαlow cells. The immediate and dramatic reduction of Srf and Myh11 mRNA expression confirmed the phenotypic change. Human rectal prolapse tissue also demonstrated significant loss of SRF expression. Conclusions SRF expression in SMCs is essential for prenatal development of the GI tract and heart. Following partial obstruction, SMCs down-regulate SRF to transition into proliferative PDGFRαlow cells that may represent a phenotype responsible for their plasticity. These findings demonstrate that SRF also plays a critical role in the remodeling process following GI injury.

Genome Sequence Variability Predicts Drug Precautions and Withdrawals from the Market

Despite substantial premarket efforts, a significant portion of approved drugs has been withdrawn from the market for safety reasons. The deleterious impact of nonsynonymous substitutions predicted by the SIFT algorithm on structure and function of drug-related proteins was evaluated for 2504 personal genomes. Both withdrawn (n = 154) and precautionary (Beers criteria (n = 90), and US FDA pharmacogenomic biomarkers (n = 96)) drugs showed significantly lower genomic deleteriousness scores (P < 0.001) compared to others (n = 752). Furthermore, the rates of drug withdrawals and precautions correlated significantly with the deleteriousness scores of the drugs (P < 0.01); this trend was confirmed for all drugs included in the withdrawal and precaution lists by the United Nations, European Medicines Agency, DrugBank, Beers criteria, and US FDA. Our findings suggest that the person-to-person genome sequence variability is a strong independent predictor of drug withdrawals and precautions. We propose novel measures of drug safety based on personal genome sequence analysis.

Serum response factor regulates smooth muscle contractility via myotonic dystrophy protein kinases and L-type calcium channels

Serum response factor (SRF) transcriptionally regulates expression of contractile genes in smooth muscle cells (SMC). Lack or decrease of SRF is directly linked to a phenotypic change of SMC, leading to hypomotility of smooth muscle in the gastrointestinal (GI) tract. However, the molecular mechanism behind SRF-induced hypomotility in GI smooth muscle is largely unknown. We describe here how SRF plays a functional role in the regulation of the SMC contractility via myotonic dystrophy protein kinase (DMPK) and L-type calcium channel CACNA1C. GI SMC expressed Dmpk and Cacna1c genes into multiple alternative transcriptional isoforms. Deficiency of SRF in SMC of Srf knockout (KO) mice led to reduction of SRF-dependent DMPK, which down-regulated the expression of CACNA1C. Reduction of CACNA1C in KO SMC not only decreased intracellular Ca2+ spikes but also disrupted their coupling between cells resulting in decreased contractility. The role of SRF in the regulation of SMC phenotype and function provides new insight into how SMC lose their contractility leading to hypomotility in pathophysiological conditions within the GI tract.

Transcriptome analysis of PDGFRα+ cells identifies T-type Ca2+ channel CACNA1G as a new pathological marker for PDGFRα+ cell hyperplasia.

Platelet-derived growth factor receptor alpha (PDGFRα)+ cells are distributed into distinct morphological groups within the serosal, muscular, and submucosal layers as well as the myenteric and deep muscular plexi. PDGFRα+ cells directly interact with interstitial cells of Cajal (ICC) and smooth muscle cells (SMC) in gastrointestinal smooth muscle tissue. These three cell types, SMC, ICC, and PDGFRα+ cells (SIP cells), form an electrical syncytium, which dynamically regulates gastrointestinal motility. We have previously reported the transcriptomes of SMC and ICC. To complete the SIP cell transcriptome project, we obtained transcriptome data from jejunal and colonic PDGFRα+ cells. The PDGFRα+ cell transcriptome data were added to the Smooth Muscle Genome Browser that we previously built for the genome-scale gene expression data of ICC and SMC. This browser provides a comprehensive reference for all transcripts expressed in SIP cells. By analyzing the transcriptomes, we have identified a unique set of PDGFRα+ cell signature genes, growth factors, transcription factors, epigenetic enzymes/regulators, receptors, protein kinases/phosphatases, and ion channels/transporters. We demonstrated that the low voltage-dependent T-type Ca2+ channel Cacna1g gene was particularly expressed in PDGFRα+ cells in the intestinal serosal layer in mice. Expression of this gene was significantly induced in the hyperplasic PDGFRα+ cells of obstructed small intestine in mice. This gene was also over-expressed in colorectal cancer, Crohn’s disease, and diverticulitis in human patients. Taken together, our data suggest that Cacna1g exclusively expressed in serosal PDGFRα+ cells is a new pathological marker for gastrointestinal diseases.