Development of a Multi-Site Phase II Clinical Trial of Valproic Acid for Retinitis Pigmentosa

The body of work presented here is a compendium of the multiple steps required for an investigator initiated trial of an existing medication (Valproic Acid- VPA) for a new indication (Retinitis Pigmentosa – RP). The chapters are listed in logical and chronological order of the process. In order to access patient records an expedited Institutional Review Board (IRB) application for retrospective chart review was submitted (Chapter 1). These records enabled the statistical analysis which not only laid the framework for the trial design, but also became the basis for two manuscripts (Chapter 2). Protocol development informed by the preliminary human studies (Chapter 3) was an instrumental part of the Investigational New Drug (IND) application (Chapter 3.5). This protocol along with the extensive case report forms that detail the intended data to be collected are included in the IND application. Because the Phase II clinical trial proposed attempting to identify the specific RP mutations of the subjects utilizing a National Eye Institute (NEI) study that enabled free genotyping services, two IRB applications were submitted (Chapter 3.6). The first was for approval of the NEI genotyping protocol, the second involved the VPA intervention. Two very different sources of funding for this trial were attempted (Chapter 4) – the NIH via the Challenge Grant mechanism and a private eye disease foundation (Foundation Fighting Blindness). In Chapter 5 I detail the alternate study designs that were considered and developed for this trial (and ultimately abandoned). Finally, in Chapter 6, I formally detail my suggestions to aid in the development of a comprehensive investigator initiated core facility at UMMMC. The goal of this project was two-fold. The first was to learn the entire process of trial and protocol design both from a Umass Institutional perspective as well as from the perspective of the FDA. The second goal was the very real prospect of helping patients with a blinding disease. This work was successful on both counts. IRB approval was received for all the submitted applications. The complexity and uniqueness of many aspects of these submissions culminated in a comprehensive learning experience. The process of working with the Umass Research Pharmacy as well as developing the industry contacts and know-how to develop a workable and financially feasible placebo were both particularly important learning experiences. FDA approval of the IND submission was also received, and the process of pre-communication and delving into the considerable and ever-changing rules and regulations resulted in an extensive and valuable knowledge base. While the practicality of funding has limited the ability of this trial to move forward at this point, given the extensive framework laid by this body of work, we are actively pursuing other opportunities. The third outcome of this work, while not as intentional, was the considerable process of determining the specific competencies and infrastructure that exist at UMMMC to enable investigator initiated drug intervention studies. While this institution is clearly moving rapidly in the direction of translational research, the many needs of these studies are often only clearly understood when the process is specifically undertaken. In completing the approval of this Phase II clinical trial, I was not only able to better understand and define the existing capabilities of UMMMC for this kind of research, I was able to add to that infrastructure when the existing knowledge or skill set was not available. In this manner, I was able to inform and guide many of the support personnel who guided me and have become a part of the strategic direction of UMMMC towards clinical translational research

Structural Association of XIST RNA with Inactive Chromosomes in Somatic Cells : a Key Step in the Process that Establishes and Faithfully Maintains X-inactivation

The XIST gene is implicated in X-chromosome inactivation, yet the RNA contains no apparent open reading frame. An accumulation of XIST RNA is observed near its site of transcription, the inactive X chromosome (Xi). A series of molecular cytogenetic studies comparing properties of XIST RNA to other protein coding RNAs, support a critical distinction for XIST RNA; XIST RNA does not concentrate at Xi simply because it is transcribed and processed there. Most notably, morphometric and 3-D analysis reveals that XIST RNA and Xi are coincident in 2-D and 3-D space; hence the XIST RNA essentially paints Xi. Several results indicate that the XIST RNA accumulation has two components, a minor one associated with transcription and processing, and a spliced major component, which stably associates with Xi. Upon transcriptional inhibition the major spliced component remains in the nucleus and often encircles the extra-prominent heterochromatic Barr body. The continually transcribed XIST gene and its poly-adenylated RNA consistently localize to a nuclear region devoid of splicing factor/poly A RNA rich domains. XIST RNA remains with the nuclear matrix fraction after removal of chromosomal DNA. XIST RNA is released from its association with Xi during mitosis, but shows a unique highly particulate distribution. Collective results indicate that XIST RNA may be an architectural element of the interphase chromosome territory, possibly a component of non-chromatin nuclear structure that specifically associates with Xi. XIST RNA is a novel nuclear RNA which potentially provides a specific precedent for RNA involvement in nuclear structure and cis-limited gene regulation via higher-order chromatin packaging

Stabilization and Localization of Xist RNA are Controlled by Separate Mechanisms and are Not Sufficient for X Inactivation

These studies address whether XIST RNA is properly localized to the X chromosome in somatic cells where human XIST expression is reactivated, but fails to result in X inactivation (Tinker, A.V., and C.J. Brown. 1998. Nucl. Acids Res. 26:2935–2940). Despite a nuclear RNA accumulation of normal abundance and stability, XIST RNA does not localize in reactivants or in naturally inactive human X chromosomes in mouse/ human hybrid cells. The XIST transcripts are fully stabilized despite their inability to localize, and hence XIST RNA localization can be uncoupled from stabilization, indicating that these are separate steps controlled by distinct mechanisms. Mouse Xist RNA tightly localized to an active X chromosome, demonstrating for the first time that the active X chromosome in somatic cells is competent to associate with Xist RNA. These results imply that species-specific factors, present even in mature, somatic cells that do not normally express Xist, are necessary for localization. When Xist RNA is properly localized to an active mouse X chromosome, X inactivation does not result. Therefore, there is not a strict correlation between Xist localization and chromatin inactivation. Moreover, expression, stabilization, and localization of Xist RNA are not sufficient for X inactivation. We hypothesize that chromosomal association of XIST RNA may initiate subsequent developmental events required to enact transcriptional silencing

A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains

BACKGROUND: Noncoding RNA species play a diverse set of roles in the eukaryotic cell. While much recent attention has focused on smaller RNA species, larger noncoding transcripts are also thought to be highly abundant in mammalian cells. To search for large noncoding RNAs that might control gene expression or mRNA metabolism, we used Affymetrix expression arrays to identify polyadenylated RNA transcripts displaying nuclear enrichment. RESULTS: This screen identified no more than three transcripts; XIST, and two unique noncoding nuclear enriched abundant transcripts (NEAT) RNAs strikingly located less than 70 kb apart on human chromosome 11: NEAT1, a noncoding RNA from the locus encoding for TncRNA, and NEAT2 (also known as MALAT-1). While the two NEAT transcripts share no significant homology with each other, each is conserved within the mammalian lineage, suggesting significant function for these noncoding RNAs. NEAT2 is extraordinarily well conserved for a noncoding RNA, more so than even XIST. Bioinformatic analyses of publicly available mouse transcriptome data support our findings from human cells as they confirm that the murine homologs of these noncoding RNAs are also nuclear enriched. RNA FISH analyses suggest that these noncoding RNAs function in mRNA metabolism as they demonstrate an intimate association of these RNA species with SC35 nuclear speckles in both human and mouse cells. These studies show that one of these transcripts, NEAT1 localizes to the periphery of such domains, whereas the neighboring transcript, NEAT2, is part of the long-sought polyadenylated component of nuclear speckles. CONCLUSION: Our genome-wide screens in two mammalian species reveal no more than three abundant large non-coding polyadenylated RNAs in the nucleus; the canonical large noncoding RNA XIST and NEAT1 and NEAT2. The function of these noncoding RNAs in mRNA metabolism is suggested by their high levels of conservation and their intimate association with SC35 splicing domains in multiple mammalian species

XIST RNA paints the inactive X chromosome at interphase: Evidence for a novel RNA involved in nuclear/chromosome structure

Abstract. The XIST gene is implicated in X chromosome inactivation, yet the RNA contains no apparent open reading frame. An accumulation of XIST RNA is observed near its site of transcription, the inactive X chromosome (Xi). A series of molecular cytogenetic studies comparing properties of XIST RNA to other protein coding RNAs, support a critical distinction for XIST RNA; XIST does not concentrate at Xi simply because it is transcribed and processed there. Most notably, morphometric and 3-D analysis reveals that XIST RNA and Xi are coincident in 2- and 3-D space; hence, the XIST RNA essentially paints Xi. Several resuits indicate that the XIST RNA accumulation has two components, a minor one associated with transcription and processing, and a spliced major component, whic

Unbalanced X;autosome translocations provide evidence for sequence specificity in the association of XIST RNA with chromatin

Whether XIST RNA is indifferent to the sequence content of the chromosome is fundamental to understanding its mechanism of chromosomal inactivation. Transgenic Xist RNA appears to associate with and inactivate an entire autosome. However, the behavior of XIST RNA on naturally occurring human X;autosome translocations has not been thoroughly investigated. Here, the relationship of human XIST RNA to autosomal chromatin is investigated in cells from two patients carrying X;autosome translocations in the context of almost complete trisomy for the involved autosome. Since trisomies of either 14 or 9 are lethal in early development, the lack of serious phenotypic consequences of the trisomy demonstrates that the translocated autosomes had been inactivated. Surprisingly, our analyses show that in primary fibroblasts from adult patients, XIST RNA does not associate with most of the involved autosome even though the bulk of it exhibits other hallmarks of inactivation beyond the region associated with XIST RNA. While results show that XIST RNA can associate with human autosomal chromatin to some degree, several observations indicate that this interaction may be unstable, with progressive loss over time. Thus, even where autosomal inactivation is selected for rather than against, there is a fundamental difference in the affinity of XIST RNA for autosomal versus X chromatin. Based on these results we propose that even autosomal chromatin that had been inactivated earlier in development may undergo a stepwise loss of inactivation hallmarks, beginning with XIST RNA. Hence compromised interaction with XIST RNA may be a primary cause of incomplete or unstable autosomal inactivation

Characterization of expression at the human XIST locus in somatic, embryonal carcinoma, and transgenic cell lines

X inactivation requires XIST, a functional RNA that is expressed exclusively from, and localizes to, the inactive X in female somatic cells. In mouse, low-level unstable transcription of Xist is observed prior to the time of inactivation, and an antisense transcript, Tsix, is a critical regulator of early Xist expression. To examine the presence and impact of an antisense transcript in humans we have characterized the extent of sense and antisense transcription in human somatic, transgenic, and embryonal carcinoma (EC) cell lines. Downstream antisense expression at the human XIST locus was not detected in somatic cells, but was detected in the EC line N-Tera2D1 and in somatic cells with an ectopic XIST locus. Presence of the antisense did not disrupt the stability or localization of the sense transcript. We have also identified additional sense transcripts in EC and female somatic cells and demonstrate that the 5\u27 flanking JPX/ENOX gene is expressed from both the active and the inactive X chromosome in somatic cell hybrids, delimiting the extent of inactive X-specific transcriptional control in somatic cells. These analyses reveal similarities to and differences from the murine Xist and Tsix transcripts and generate a complex picture of developmentally regulated transcription through the region

An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles

NEAT1 RNA, a highly abundant 4 kb ncRNA, is retained in nuclei in approximately 10 to 20 large foci that we show are completely coincident with paraspeckles, nuclear domains implicated in mRNA nuclear retention. Depletion of NEAT1 RNA via RNAi eradicates paraspeckles, suggesting that it controls sequestration of the paraspeckle proteins PSP1 and p54, factors linked to A-I editing. Unlike overexpression of PSP1, NEAT1 overexpression increases paraspeckle number, and paraspeckles emanate exclusively from the NEAT1 transcription site. The PSP-1 RNA binding domain is required for its colocalization with NEAT1 RNA in paraspeckles, and biochemical analyses support that NEAT1 RNA binds with paraspeckle proteins. Unlike other nuclear-retained RNAs, NEAT1 RNA is not A-I edited, consistent with a structural role in paraspeckles. Collectively, results demonstrate that NEAT1 functions as an essential structural determinant of paraspeckles, providing a precedent for a ncRNA as the foundation of a nuclear domain