Brendan Lee

Genetic testing has undergone a revolution in the last decade, particularly with the advent of next-generation sequencing and its associated reductions in costs and increases in efficiencies. The Undiagnosed Diseases Network (UDN) has been a leader in the application of such genomic testing for rare disease diagnosis. This review discusses the current state of genomic testing performed within the UDN, with a focus on the strengths and limitations of whole-exome and whole-genome sequencing in clinical diagnostics and the importance of ongoing data reanalysis. The role of emerging technologies such as RNA and long-read sequencing to further improve diagnostic rates in the UDN is also described. This review concludes with a discussion of the challenges faced in insurance coverage of comprehensive genomic testing as well as the opportunities for a larger role of testing in clinical medicine.

Osteoarthritis (OA) is a common degenerative condition that afflicts more than 70% of the population between 55 and 77 years of age. Although its prevalence is rising globally with aging of the population, current therapy is limited to symptomatic relief and, in severe cases, joint replacement surgery. We report that intra-articular expression of proteoglycan 4 (Prg4) in mice protects against development of OA. Long-term Prg4 expression under the type II collagen promoter (Col2a1) does not adversely affect skeletal development but protects from developing signs of age-related OA. The protective effect is also shown in a model of posttraumatic OA created by cruciate ligament transection. Moreover, intra-articular injection of helper-dependent adenoviral vector expressing Prg4 protected against the development of posttraumatic OA when administered either before or after injury. Gene expression profiling of mouse articular cartilage and in vitro cell studies show that Prg4 expression inhibits the transcriptional programs that promote cartilage catabolism and hypertrophy through the up-regulation of hypoxia-inducible factor 3α. Analyses of available human OA data sets are consistent with the predictions of this model. Hence, our data provide insight into the mechanisms for OA development and offer a potential chondroprotective approach to its treatment.

Objective: The mouse is an optimal model organism in which gene–environment interactions can be used to study the pathogenesis of osteoarthritis (OA). The gold standard for arthritis research in mice is based on histopathology and immunohistochemistry, which are labor‐intensive, prone to sampling bias and technical variability, and limited in throughput. The aim of this study was to develop a new technique that assesses mouse cartilage by integrating quantitative volumetric imaging techniques.Methods: A novel mouse model of OA was generated by cruciate ligament transection (CLT) and evaluated by histopathology and immunohistochemistry. Knee joint specimens were then imaged using a new technique that combines high‐resolution micro–computed tomography (micro‐CT) and phase‐contrast optics followed by quantitative analyses. A comparative analysis was also performed in a previously established mouse model of OA generated by destabilization of the medial meniscus (DMM).Results: Phase‐contrast micro‐CT achieved cellular resolution of chondrocytes and quantitative assessment of parameters such as articular cartilage volume and surface area. In mouse models of OA generated by either CLT or DMM, we showed that phase‐contrast micro‐CT distinguished control and OA cartilage by providing quantitative measures with high reproducibility and minimal variability. Features of OA at the cellular or tissue level could also be observed in images generated by phase‐contrast micro‐CT.Conclusion: We established an imaging technology that comprehensively assessed and quantified the 2‐dimensional and 3‐dimensional changes of articular cartilage. Application of this technology will facilitate the rapid and high‐throughput assessment of genetic and therapeutic models of OA in mice.

A major obstacle in the genetic therapy of inherited metabolic disease is host immune responses to the therapeutic protein. This is best exemplified by inhibitor formation in the protein therapy for hemophilia A. An approach to overcoming this is induction of immunological tolerance to the therapeutic protein. Tolerogenic dendritic cells (DCtols) have been reported to induce tolerance. In addition, cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β ₁ are known to induce tolerance. To model protein therapy, we used ovalbumin (OVA) as antigen in BALB/c mice and their transgenic derivative, DO11.10 mice. In this study we show that adoptive transfer of antigen-pulsed dendritic cells (DCs) treated with a combination of IL-10 and TGF-β ₁ can suppress the antibody response in mice. Adoptive transfer of cytokine-conditioned DCs in preimmunized mice results in reduction of antibody response in the mice. Furthermore, the effect is antigen specific, as the recipient mice were able to mount a potent antibody response to the control antigen. Last, we show that TGF-β ₁ and IL-10-conditioned DCs are able to inhibit anti-FVIII antibody responses in FVIII knockout (KO) mice. Analysis of the contribution of IL-10 and TGF-β ₁ to the DCtol phenotype shows that IL-10 treatment of DCs is sufficient for inducing OVA-specific tolerance in BALB/c mice, but we observed a requirement for treatment with both human TGF-β ₁ and human IL-10 to significantly inhibit anti-FVIII antibody responses in FVIII KO mice. This paper demonstrates that autologous cell therapy for antigen-targeted immune suppression may be developed to facilitate long-term therapy.

We previously demonstrated that Toll-like receptor/myeloid differentiation primary response gene 88 (MyD88) signaling is required for maximal innate and acquired [T helper cell type 1 (Th1)] immune responses following systemic administration of helper-dependent adenoviral vectors (HDAds). However, MyD88-deficient mice injected with HDAdLacZ exhibited only partial reduction of innate immune cytokine expression compared with wild-type mice, suggesting MyD88-independent pathways also respond to HDAds. We now show that NOD2, a nucleotide-binding and oligomerization domain (NOD)–like receptor known to detect muramyl dipeptides in bacterial peptidoglycans, also contributes to innate responses to HDAds, but not to humoral or Th1 immune responses. We established NOD2/MyD88 double-deficient mice that, when challenged with HDAds, showed a significant reduction of the innate response compared with mice deficient for either gene singly, suggesting that NOD2 signaling contributes to the innate response independently of MyD88 signaling following systemic administration of HDAds. In addition, NOD2-deficient mice exhibited significantly higher transgene expression than did wild-type mice at an early time point (before development of an acquired response), but not at a later time point (after development of an acquired response). These results indicate that the intracellular sensor NOD2 is required for innate responses to HDAds and can limit transgene expression during early phases of infection.

TGFβ and BMP signaling pathways exhibit antagonistic activities during the development of many tissues. Although the crosstalk between BMP and TGFβ signaling pathways is well established in bone development, the relationship between these two pathways is less well defined during cartilage development and postnatal homeostasis. We generated hypomorphic mouse models of cartilage-specific loss of BMP and TGFβ signaling to assess the interaction of these pathways in postnatal growth plate homeostasis. We further used the chondrogenic ATDC5 cell line to test effects of BMP and TGFβ signaling on each other's downstream targets. We found that conditional deletion of Smad1 in chondrocytes resulted in a shortening of the growth plate. The addition of Smad5 haploinsufficiency led to a more severe phenotype with shorter prehypertrophic and hypertrophic zones and decreased chondrocyte proliferation. The opposite growth plate phenotype was observed in a transgenic mouse model of decreased chondrocytic TGFβ signaling that was generated by expressing a dominant negative form of the TGFβ receptor I (ΔTβRI) in cartilage. Histological analysis demonstrated elongated growth plates with enhanced Ihh expression, as well as an increased proliferation rate with altered production of extracellular matrix components. In contrast, in chondrogenic ATDC5 cells, TGFβ was able to enhance BMP signaling, while BMP2 significantly reduces levels of TGF signaling. In summary, our data demonstrate that during endochondral ossification, BMP and TGFβ signaling can have antagonistic effects on chondrocyte proliferation and differentiation in vivo. We also found evidence of direct interaction between the two signaling pathways in a cell model of chondrogenesis in vitro.

Gene therapy for genetic diseases has entered into its third decade since the first human clinical trial in 1990. As such it has reached young adulthood with real achievements and great promise that befits this period in life. This review issue highlights the advances in both the preclinical and clinical arenas, successes long in coming to the field, and the obstacles that still remain. While the concept of gene therapy was enunciated formally in the 70s, the first in vitro gene transfer proof of concept studies were not widely performed until the 80s. In 1990, the first human gene transfer clinical trial was performed in patients with adenosine deaminase deficiency. This landmark achievement has now ballooned to over 1700 worldwide clinical trials involving cancer, infectious disease, Mendelian genetic diseases, and complex common diseases (Journal of Gene Medicine, http://www.abedia.com/wiley/index.html). The vast majority are phase I clinical trials (60%), followed by phase I/II and II clinical trials (35%), with a minority being phase II/III and III clinical trials (<5%). During this time, there has been a proliferation of both viral and nonviral gene transfer vectors. The majority of viral gene transfer trials have involved adenovirus and retroviruses, though adeno-associated virus and lentivirus vectors have become major foci for both clinical and preclinical study. Not surprisingly, 65% of human clinical trials were performed in the context of cancer. While the risk/benefit considerations in cancer make it the logical target for novel biological therapies, the common complex genetic diseases constitute an enormous target population. However, because of the significant advances in understanding the basic pathophysiology of Mendelian disorders, these have often been the arena for initial development of vectors and preclinical studies. Still, the progression to clinical utility has been frustrating though recent breakthroughs have re-energized the field as a whole.