Nonclinical Safety Evaluation of scAAV8-RLBP1 for Treatment of RLBP1 Retinitis Pigmentosa.

Retinitis pigmentosa is a form of retinal degeneration usually caused by genetic mutations affecting key functional proteins. We have previously demonstrated efficacy in a mouse model of RLBP1 deficiency with a self-complementary AAV8 vector carrying the gene for human RLBP1 under control of a short RLBP1 promoter (CPK850).1 In this article, we describe the nonclinical safety profile of this construct as well as updated efficacy data in the intended clinical formulation. In Rlbp1-/- mice dosed at a range of CPK850 levels, a minimum efficacious dose of 3 × 107 vg in a volume of 1 µL was observed. For safety assessment in these and Rlbp1+/+ mice, optical coherence tomography (OCT) and histopathological analysis indicated retinal thinning that appeared to be dose-dependent for both Rlbp1 genotypes, with no qualitative difference noted between Rlbp1+/+ and Rlbp1-/- mice. In a non-human primate study, RLBP1 mRNA expression was detected and dose dependent intraocular inflammation and retinal thinning were observed. Inflammation resolved slowly over time and did not appear to be exacerbated in the presence of anti-AAV8 antibodies. Biodistribution was evaluated in rats and satellite animals in the non-human primate study. The vector was largely detected in ocular tissues and low levels in the optic nerve, superior colliculus, and lateral geniculate nucleus, with limited distribution outside of these tissues. These data suggest that an initial subretinal dose of ∼3 × 107 vg/µL CPK850 can safely be used in clinical trials.

Block of C/EBP alpha function by phosphorylation in acute myeloid leukemia with FLT3 activating mutations.

Mutations constitutively activating FLT3 kinase are detected in approximately 30% of acute myelogenous leukemia (AML) patients and affect downstream pathways such as extracellular signal-regulated kinase (ERK)1/2. We found that activation of FLT3 in human AML inhibits CCAAT/enhancer binding protein alpha (C/EBPalpha) function by ERK1/2-mediated phosphorylation, which may explain the differentiation block of leukemic blasts. In MV4;11 cells, pharmacological inhibition of either FLT3 or MEK1 leads to granulocytic differentiation. Differentiation of MV4;11 cells was also observed when C/EBPalpha mutated at serine 21 to alanine (S21A) was stably expressed. In contrast, there was no effect when serine 21 was mutated to aspartate (S21D), which mimics phosphorylation of C/EBPalpha. Thus, our results suggest that therapies targeting the MEK/ERK cascade or development of protein therapies based on transduction of constitutively active C/EBPalpha may prove effective in treatment of FLT3 mutant leukemias resistant to the FLT3 inhibitor therapies.

PLZF is a negative regulator of retinoic acid receptor transcriptional activity.

BACKGROUND: Retinoic acid receptors (RARs) are ligand-regulated transcription factors controlling cellular proliferation and differentiation. Receptor-interacting proteins such as corepressors and coactivators play a crucial role in specifying the overall transcriptional activity of the receptor in response to ligand treatment. Little is known however on how receptor activity is controlled by intermediary factors which interact with RARs in a ligand-independent manner. RESULTS: We have identified the promyelocytic leukemia zinc finger protein (PLZF), a transcriptional corepressor, to be a RAR-interacting protein using the yeast two-hybrid assay. We confirmed this interaction by GST-pull down assays and show that the PLZF N-terminal zinc finger domain is necessary and sufficient for PLZF to bind RAR. The RAR ligand binding domain displayed the highest affinity for PLZF, but corepressor and coactivator binding interfaces did not contribute to PLZF recruitment. The interaction was ligand-independent and correlated to a decreased transcriptional activity of the RXR-RAR heterodimer upon overexpression of PLZF. A similar transcriptional interference could be observed with the estrogen receptor alpha and the glucocorticoid receptor. We further show that PLZF is likely to act by preventing RXR-RAR heterodimerization, both in-vitro and in intact cells. CONCLUSION: Thus RAR and PLZF interact physically and functionally. Intriguingly, these two transcription factors play a determining role in hematopoiesis and regionalization of the hindbrain and may, upon chromosomal translocation, form fusion proteins. Our observations therefore define a novel mechanism by which RARs activity may be controlled.

Morphogenesis of the telencephalic commissure requires scaffold protein JNK-interacting protein 3 (JIP3).

The murine JNK-interacting protein 3 (JIP3) protein (also known as JSAP1) is expressed exclusively in neurons and has been identified as a scaffold protein for the c-Jun NH2-terminal kinase (JNK) signaling pathway and as an adapter protein for cargo transport by the microtubule motor protein kinesin. To investigate the physiological function of JIP3, we examined the effect of Jip3 gene disruption in mice. The Jip3-/- mice were unable to breathe and died shortly after birth. Microscopic analysis demonstrated that Jip3 gene disruption causes severe defects in the morphogenesis of the telencephalon. Jip3-/- mice lack the telencephalic commissure, a major connection between the left and right hemispheres of the brain. The central nervous system abnormalities of Jip3-/- mice may be accounted for in part by a reduction in signal transduction by RhoA and its effector ROCK.

Retinoic acid receptors inhibit AP1 activation by regulating extracellular signal-regulated kinase and CBP recruitment to an AP1-responsive promoter.

Retinoids exhibit antineoplastic activities that may be linked to retinoid receptor-mediated transrepression of activating protein 1 (AP1), a heterodimeric transcription factor composed of fos- and jun-related proteins. Here we show that transcriptional activation of an AP1-regulated gene through the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway (MAPK(ERK)) is characterized, in intact cells, by a switch from a fra2-junD dimer to a junD-fosB dimer loading on its promoter and by simultaneous recruitment of ERKs, CREB-binding protein (CBP), and RNA polymerase II. All-trans-retinoic acid (atRA) receptor (RAR) was tethered constitutively to the AP1 promoter. AP1 transrepression by retinoic acid was concomitant to glycogen synthase kinase 3 activation, negative regulation of junD hyperphosphorylation, and to decreased RNA polymerase II recruitment. Under these conditions, fra1 loading to the AP1 response element was strongly increased. Importantly, CBP and ERKs were excluded from the promoter in the presence of atRA. AP1 transrepression by retinoids was RAR and ligand dependent, but none of the functions required for RAR-mediated transactivation was necessary for AP1 transrepression. These results indicate that transrepressive effects of retinoids are mediated through a mechanism unrelated to transcriptional activation, involving the RAR-dependent control of transcription factors and cofactor assembly on AP1-regulated promoters.