Therapeutic targeting of membrane-associated GRP78 in leukemia and lymphoma: preclinical efficacy in vitro and formal toxicity study of BMTP-78 in rodents and primates.

Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D(KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.

An AAVP-based solid-phase transducing matrix for transgene delivery: potential for translational applications.

A hybrid vector of adeno-associated virus and phage (termed AAVP) has been introduced as a platform for systemic ligand-directed delivery of transgenes to tumors over the past decade. A series of studies have evaluated the AAVP platform for potential theranostic or purely therapeutic applications in several tumor models. Sufficient ligand-directed tumor targeting consistently resulted in specific molecular-genetic imaging and/or anti-tumor responses to 'suicide' transgene delivery. However, efforts to optimize transduction efficiency are still ongoing. Here, we set out to expand the translational utility of AAVP by combining it with gold (Au) nanoparticles in order to generate a 'transducing matrix' for improved targeted gene delivery in solid phase. Targeted AAVP-based solid-phase transduction is superior to conventional transduction in soluble (aqueous) environments. This transducing matrix is stable and can be further modified with additional attributes (for example, magnetization) for targeted imaging and therapeutic gene delivery. Notably, it spontaneously assembles around cells in vitro to markedly enhance transduction capabilities compared with AAVP alone. This versatile nanoplatform may enable new applications of AAVP for transgene delivery in translational settings including, for example, efforts toward complex tissue patterning.

Magnetic resonance imaging and micro-computed tomography combined atlas of developing and adult mouse brains for stereotaxic surgery.

Stereotaxic atlases of the mouse brain are important in neuroscience research for targeting of specific internal brain structures during surgical operations. The effectiveness of stereotaxic surgery depends on accurate mapping of the brain structures relative to landmarks on the skull. During postnatal development in the mouse, rapid growth-related changes in the brain occur concurrently with growth of bony plates at the cranial sutures, therefore adult mouse brain atlases cannot be used to precisely guide stereotaxis in developing brains. In this study, three-dimensional stereotaxic atlases of C57BL/6J mouse brains at six postnatal developmental stages: postnatal day (P) 7, P14, P21, P28, P63 and in adults (P140-P160) were developed, using diffusion tensor imaging (DTI) and micro-computed tomography (CT). At present, most widely-used stereotaxic atlases of the mouse brain are based on histology, but the anatomical fidelity of ex vivo atlases to in vivo mouse brains has not been evaluated previously. To account for ex vivo tissue distortion due to fixation as well as individual variability in the brain, we developed a population-averaged in vivo magnetic resonance imaging adult mouse brain stereotaxic atlas, and a distortion-corrected DTI atlas was generated by nonlinearly warping ex vivo data to the population-averaged in vivo atlas. These atlas resources were developed and made available through a new software user-interface with the objective of improving the accuracy of targeting brain structures during stereotaxic surgery in developing and adult C57BL/6J mouse brains.

Persistent hypersynchronization of neocortical neurons in the mocha mutant of mouse.

A recessive mutation in the mouse at the mocha locus (mh, chromosome 10) modulates the synchronous synaptic activation of neocortical neurons, resulting in a constant 6-7 Hz (theta) wave pattern in the electrocorticogram. The gene-linked brain rhythm is unaffected by motor behavior and cannot be desynchronized by sensory stimuli. This exemplary neurological mutation affecting cortical excitability is the first to reveal clearly that the predominance of a specific pattern of spontaneous brain wave activity can be inherited as a recessive trait.

p21 controls patterning but not homologous recombination in RPE development.

p21/WAF1/CIP1/MDA6 is a key cell cycle regulator. Cell cycle regulation is an important part of development, differentiation, DNA repair and apoptosis. Following DNA damage, p53 dependent expression of p21 results in a rapid cell cycle arrest. p21 also appears to be important for the development of melanocytes, promoting their differentiation and melanogenesis. Here, we examine the effect of p21 deficiency on the development of another pigmented tissue, the retinal pigment epithelium. The murine mutation pink-eyed unstable (p(un)) spontaneously reverts to a wild-type allele by homologous recombination. In a retinal pigment epithelium cell this results in pigmentation, which can be observed in the adult eye. The clonal expansion of such cells during development has provided insight into the pattern of retinal pigment epithelium development. In contrast to previous results with Atm, p53 and Gadd45, p(un) reversion events in p21 deficient mice did not show any significant change. These results suggest that p21 does not play any role in maintaining overall genomic stability by regulating homologous recombination frequencies during development. However, the absence of p21 caused a distinct change in the positions of the reversion events within the retinal pigment epithelium. Those events that would normally arrest to produce single cell events continued to proliferate uncovering a cell cycle dysregulation phenotype. It is likely that p21 is involved in controlling the developmental pattern of the retinal pigment. We also found a C57BL/6J specific p21 dependent ocular defect in retinal folding, similar to those reported in the absence of p53.

Spare the rod and spoil the eye.

This review presents a new unified view of the pathogenesis of three common causes of acquired retinal degenerative disease-diabetic retinopathy, age related macular degeneration, and retinopathy of prematurity. In these three conditions, angiogenesis has a predominant role in the development of sight threatening pathology. Angiogenesis is controlled by among other factors the expression of vascular endothelial growth factor (VEGF), which in turn is regulated by absolute and relative lack of oxygen. The severe pathological manifestations of these three conditions are not part of a general underlying disease process because they are peculiar to the eye, and the profound hypoxia that develops in normal retina during dark adaptation (rod driven hypoxia) is an adequate and elegant additional factor to explain their pathogenesis. A large number of experimental reports support this conclusion, although rod driven anoxia is not generally considered as a causal factor in ocular disease. However, the hypothesis can be critically tested, and also suggests novel methods of treatment and prevention of these conditions that may be simpler and more inexpensive than current therapies and that have a smaller potential for adverse effects.

An anti-angiogenic state in mice and humans with retinal photoreceptor cell degeneration.

Abnormal angiogenesis accompanies many pathological conditions including cancer, inflammation, and eye diseases. Proliferative retinopathy because of retinal neovascularization is a leading cause of blindness in developed countries. Another major cause of irreversible vision loss is retinitis pigmentosa, a group of diseases characterized by progressive photoreceptor cell degeneration. Interestingly, anecdotal evidence has long suggested that proliferative diabetic retinopathy is rarely associated clinically with retinitis pigmentosa. Here we show that neonatal mice with classic inherited retinal degeneration (Pdeb(rd1)/Pdeb(rd1)) fail to mount reactive retinal neovascularization in a mouse model of oxygen-induced proliferative retinopathy. We also present a comparable human paradigm: spontaneous regression of retinal neovascularization associated with long-standing diabetes mellitus occurs when retinitis pigmentosa becomes clinically evident. Both mouse and human data indicate that reactive retinal neovascularization either fails to develop or regresses when the number of photoreceptor cells is markedly reduced. Our findings support the hypothesis that a functional mechanism underlying this anti-angiogenic state is failure of the predicted up-regulation of vascular endothelial growth factor, although other growth factors may also be involved. Preventive and therapeutic strategies against both proliferative and degenerative retinopathies may emerge from this work.

Segregation of human neural stem cells in the developing primate forebrain.

Many central nervous system regions at all stages of life contain neural stem cells (NSCs). We explored how these disparate NSC pools might emerge. A traceable clone of human NSCs was implanted intraventricularly to allow its integration into cerebral germinal zones of Old World monkey fetuses. The NSCs distributed into two subpopulations: One contributed to corticogenesis by migrating along radial glia to temporally appropriate layers of the cortical plate and differentiating into lamina-appropriate neurons or glia; the other remained undifferentiated and contributed to a secondary germinal zone (the subventricular zone) with occasional members interspersed throughout brain parenchyma. An early neurogenetic program allocates the progeny of NSCs either immediately for organogenesis or to undifferentiated pools for later use in the "postdevelopmental" brain.

Susceptibility of proliferating cells to benzo[a]pyrene-induced homologous recombination in mice.

The pink-eyed unstable mutation, p(un), is the result of a 70 kb tandem duplication within the murine pink-eyed, p, gene. Deletion of one copy of the duplicated region by homologous deletion/recombination occurs spontaneously in embryos and results in pigmented spots in the fur and eye. Such deletion events are inducible by a variety of DNA damaging agents, as we have observed previously with both fur- and eye-spot assays. Here we describe a study of the effect of exposure to benzo[a]pyrene (B[a]P) at different times of development on reversion induction in the eye. Previously we, among others, have reported that the retinal pigment epithelium (RPE) displays a position effect variegation phenotype in the pattern of pink-eyed unstable reversions. Following an acute exposure to B[a]P or X-rays on the tenth day of gestation an increased frequency of reversion events was detected in a distinct region of the adult RPE. Examining exposure at different times of eye development reveals that both B[a]P and X-rays result in an increased frequency of reversion events, though the increase was only significant following B[a]P exposure, similar to our previous report limited to exposure on the tenth day of gestation. Examination of B[a]P-exposed RPE in the present study revealed distinct regions where the induced events lie and that the positions of these regions are found at increasing distances from the optic nerve the later the time of exposure. This position effect directly reflects the previously observed developmental pattern of the RPE, namely that cells in the regions most distal from the optic nerve are proliferating most vigorously. The numbers and positions of RPE cells displaying the transformed (pigmented) phenotype strongly advocate the proposal that dividing cells are at highest risk to deletions induced by carcinogens.

Benzo(a)pyrene and X-rays induce reversions of the pink-eyed unstable mutation in the retinal pigment epithelium of mice.

The pink-eyed unstable (p(un)) mutation is the result of a 70kb tandem duplication within the murine p gene. Homologous deletion/recombination of the locus to wild-type occurs spontaneously in embryos and results in pigmented spots in the fur and eye that persist for life. Such deletion events are also inducible by a variety of DNA damaging agents, as we have observed previously with the fur spot assay. Here, we describe the use of the retinal pigment epithelium (RPE) of the eye to detect reversion events induced with two differently acting agents. Benzo(a)pyrene (B(a)P) induces a high frequency, and X-ray exposure a more modest increase, of p(un) reversion in both the fur and the eye. The eye-spot assay requires fewer mice for significant results than the fur spot assay. Previous work had elucidated the cell proliferation pattern in the RPE and a position effect variegation phenotype in the pattern of p(un) reversions, which we have confirmed. Acute exposure to B(a)P or X-rays resulted in an increased frequency of reversion events. The majority of the spontaneous reversions lie toward the periphery of the RPE whereas induced events are found more centrally, closer to the optic nerve head. The induced distribution corresponds to the major sites of cell proliferation in the RPE at the time of exposure, and further advocates the proposal that dividing cells are at highest risk to develop deletions.

Phototransduction in transgenic mice after targeted deletion of the rod transducin alpha -subunit.

Retinal photoreceptors use the heterotrimeric G protein transducin to couple rhodopsin to a biochemical cascade that underlies the electrical photoresponse. Several isoforms of each transducin subunit are present in the retina. Although rods and cones seem to contain distinct transducin subunits, it is not known whether phototransduction in a given cell type depends strictly on a single form of each subunit. To approach this question, we have deleted the gene for the rod transducin alpha-subunit in mice. In hemizygous knockout mice, there was a small reduction in retinal transducin alpha-subunit content but retinal morphology and the physiology of single rods were largely normal. In homozygous knockout mice, a mild retinal degeneration occurred with age. Rod-driven components were absent from the electroretinogram, whereas cone-driven components were retained. Every photoreceptor examined by single-cell recording failed to respond to flashes, with one exception. The solitary responsive cell was insensitive, as expected for a cone, but had a rod-like spectral sensitivity and flash response kinetics that were slow, even for rods. These results indicate that most if not all rods use a single transducin type in phototransduction.

Morphological, physiological, and biochemical changes in rhodopsin knockout mice.

Mutations in rod opsin, the visual pigment protein of rod photoreceptors, account for approximately 15% of all inherited human retinal degenerations. However, the physiological and molecular events underlying the disease process are not well understood. One approach to this question has been to study transgenic mice expressing opsin genes containing defined mutations. A caveat of this approach is that even the overexpression of normal opsin leads to photoreceptor cell degeneration. To overcome the problem, we have reduced or eliminated endogenous rod opsin content by targeted gene disruption. Retinas in mice lacking both opsin alleles initially developed normally, except that rod outer segments failed to form. Within months of birth, photoreceptor cells degenerated completely. Retinas from mice with a single copy of the opsin gene developed normally, and rods elaborated outer segments of normal size but with half the normal complement of rhodopsin. Photoreceptor cells in these retinas also degenerated but did so over a much slower time course. Physiological and biochemical experiments showed that rods from mice with a single opsin gene were approximately 50% less sensitive to light, had accelerated flash-response kinetics, and contained approximately 50% more phosducin than wild-type controls.

Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes.

Stable clones of neural stem cells (NSCs) have been isolated from the human fetal telencephalon. These self-renewing clones give rise to all fundamental neural lineages in vitro. Following transplantation into germinal zones of the newborn mouse brain they participate in aspects of normal development, including migration along established migratory pathways to disseminated central nervous system regions, differentiation into multiple developmentally and regionally appropriate cell types, and nondisruptive interspersion with host progenitors and their progeny. These human NSCs can be genetically engineered and are capable of expressing foreign transgenes in vivo. Supporting their gene therapy potential, secretory products from NSCs can correct a prototypical genetic metabolic defect in neurons and glia in vitro. The human NSCs can also replace specific deficient neuronal populations. Cryopreservable human NSCs may be propagated by both epigenetic and genetic means that are comparably safe and effective. By analogy to rodent NSCs, these observations may allow the development of NSC transplantation for a range of disorders.

Differentiation of engrafted multipotent neural progenitors towards replacement of missing granule neurons in meander tail cerebellum may help determine the locus of mutant gene action.

Previously we observed that stable clones of multipotent neural progenitor cells, initially isolated and propagated from the external granular layer of newborn wild-type mouse cerebellum, could participate appropriately in cerebellar development when reimplanted into the external granular layer of normal mice. Donor cells could reintegrate and differentiate into neurons (including granule cells) and/or glia consistent with their site of engraftment. These findings suggested that progenitors might be useful for cellular replacement in models of aberrant neural development or neurodegeneration. We tested this hypothesis by implanting clonally related multipotent progenitors into the external granular layer of newborn meander tail mice (gene symbol=mea). mea is an autosomal recessive mutation characterized principally by the failure of granule cells to develop in the cerebellar anterior lobe; the mechanism is unknown. We report that approximately 75% of progenitors transplanted into the granuloprival anterior lobe of neonatal mea mutants differentiated into granule cells, partially replacing or augmenting that largely absent neuronal population in the internal granular layer of the mature meander tail anterior lobe. (The ostensibly 'normal' meander tail posterior lobe also benefited from repletion of a more subtle granule cell deficiency.) Donor-derived neurons were well-integrated within the neuropil, suggesting that these progenitors' developmental programs for granule cell differentiation were unperturbed. These observations permitted several conclusions. (1) That exogenous progenitors could survive transplantation into affected regions of neonatal meander tail cerebellum and differentiate into the deficient cell type suggested that the microenvironment was not inimical to granule cell development. Rather it suggested that mea's deleterious action is intrinsic to the external granular layer cell. (Any cell-extrinsic actions--albeit unlikely--had to be restricted to readily circumventable prenatal events.) This study, therefore, offers a paradigm for using progenitors to help determine the site of action of other mutant genes or to test hypotheses regarding the pathophysiology underlying other anomalies. (2) In the regions most deficient in neurons, a neuronal phenotype was pursued in preference to other potential cell types, suggesting a 'push' of undifferentiated, multipotent progenitors towards compensation for granule cell dearth. These data suggested that progenitors with the potential for multiple fates might differentiate towards repletion of deficient cell types, a possible developmental mechanism with therapeutic implications. Neural progenitors (donor or endogenous) might enable cell replacement in some developmental or degenerative diseases--most obviously in cases where a defect is intrinsic to the diseased cell, but also, under certain circumstances, when extrinsic pathologic forces may exist.

ERG abnormalities in relation to histopathologic findings in vitiligo mutant mice.

The vitiligo, mivit, mutation has several prenatal and perinatal effects on development of the retinal pigment epithelium, and later, leads to extensive, progressive degeneration of photoreceptor cells in the neural retina of homozygous affected mice. The aim of the present study was to determine by functional criteria how early can abnormalities be detected in the neural retina. Electroretinograms (ERGs) were correlated with histopathological findings in the same animals. Congenic homozygous mutants, heterozygotes, and homozygous wild-type mice were studied at 2, 3, 6, 24 and 56 weeks of age, the same animals being tested serially at the three older time points. The nontested eye of each animal was embedded in Epon and sectioned at 1 micron for light microscopic study. ERG recordings from vitiligo homozygotes differed from heterozygous and wild-type mice, but the latter two groups did not differ from each other. As early as two weeks of age, homozygous mutants showed a significant reduction of rod dominated maximum ERG a-wave and b-wave amplitude. ERG b-wave sensitivity (sigma) was significantly reduced, and ERG implicit times were delayed for homozygous mutants at 3 (a-wave) and 6 (b-wave) weeks of age. This is the first study to report reduced and delayed ERG a-waves and b-waves in this animal model, like the early functional abnormalities in human retinitis pigmentosa, and also the first to show short and disoriented rod outer segments, beginning retinal separation from the pigment epithelium, and a few macrophage-like cells already present in the subretinal space at 2 weeks of age (in three of four homozygous mutant eyes examined). Given these early functional and structural abnormalities in the neural retina, it remains to be determined whether the mi gene targets the retinal pigment epithelial cell, the photoreceptor cell, or both.

Mapping and retinal phenotype of the hugger mutation in the mouse.

Hugger, hug, is a recessively expressed mutation in mice that features mildly abnormal locomotion, not yet explained, and a unique combination of developmental and degenerative retinal abnormalities. Analysis with the efficient MEV linkage testing stock established that hug is on mouse Chr 19 about 14 cM from th centromere, between the microsatellite markers D19Mit28 and D19Mit14. An abnormal retinal phenotype was recognized on the day of birth, when some retinal ganglion cells already lie in abnormal positions in the inner plexiform layer. By postnatal day 18 the number of neurons is reduced in all three cellular layers of the retina. Rod photoreceptor cells develop only rudimentory outer segments, and by 9 months of age, about 75% of the photoreceptor cells have completely disappeared. Similar photoreceptor cell abnormalities are seen in prph2 (formerly rds) homozygotes, which lack the peripherin/rds protein of the rod outer segments, but a mating of the respective homozygotes yielded normal progeny. Rom1, which codes for an outer segment protein similar to peripherin/rds, maps to a more proximal position on Chr 19.

Phagosome number and distribution in retinal pigment epithelial cells of vitiligo mutant mice.

The vitiligo mutant mouse has a disorder affecting the interaction of retinal pigment epithelium (RPE) and photoreceptor cells of the neural retina. Among the phenotypic features are patches of hyper- and hypopigmentation in the embryonic RPE, increased RPE cell production neonatally, and a later onset of progressive photoreceptor cell degeneration that continues for more than one year until all photoreceptor cells are gone. Failure of RPE microvilli to intertwine with rod outer segments (ROS) at any age, the accumulation of ROS membranous fragments in the subretinal space, and a relatively early retinal separation from the RPE suggested analysis of whether RPE phagocytosis might be impaired. Post-natal day 23 (P23) and P36 mutant and congenic control wild-type mice were kept in darkness overnight and eyes were examined by light and transmission electron microscopy 0.5 hr before, 1.5 hr after and 10.5 hr after lights turned on at 0700 hr. At these ages ROS have not yet degenerated, though they are shorter than normal and somewhat misoriented. The number of phagosomes per RPE cell was markedly reduced in mutants compared to controls at both ages and all time points. Nonetheless, the highest counts were obtained 1.5 hr after the lights turned on in mutant and control specimens. In the mutant eyes, the proportion of phagosomes in the microvillous zone of the RPE cells was consistently lower than in any other cellular compartment. Phagosome distribution in the apical and basal zones of the RPE cell cytoplasm was within normal limits. Macrophage-like cells become numerous in the subretinal space at older ages, but were already present at P23 and P36, and contained phagosomes in their cytoplasm. The hypothesis is proposed that binding of ROS to RPE cells might be defective in vitiligo mice, in contrast to the rdy rat, where the work of others indicates that binding is normal and the subsequent ingestion of phagosomes is impaired.

Pigment epithelial and retinal phenotypes in the vitiligo mivit, mutant mouse.

PURPOSE: To describe the abnormal phenotype in retinal pigment epithelium (RPE) and neural retina of vitiligo mutant mice from embryonic stages to old age. METHODS: Eyes of wild-type controls and congenic vitiligo mutants were examined by light and electron microscopy from embryonic day (E) 12 to 2 years of age. The amount and distribution of pigment in the RPE was studied in wholemounts. RESULTS: Earliest phenotypic expression of mivit is seen in the RPE, which is abnormally multilayered dorsally at E12 to E13, and contains both hyperpigmented and hypopigmented patches. Postnatally, most RPE cells have abnormally short, compact, apical microvilli not containing melanosomes and not interdigitating with rod outer segments (ROS). Rod outer segments begin to degenerate relatively late, at approximately postnatal day (P) 30, and fragments accumulate in the subretinal space; photoreceptor nuclei decrease in number progressively from approximately P60 to P500. Retinal detachment, more prominent than in most other retinal degenerations, begins as ROS break up. Additional unusual events are the appearance of macrophage-like cells in the subretinal space by P21 to P60 and extensive shedding of photoreceptor nuclei across the external limiting membrane and into the subretinal space from approximately P180 to P500. Photoreceptor cell degeneration follows a radial gradient, more severe centrally, and is more advanced superiorly than inferiorly. By 2 years, almost all rod and cone cells are gone, and the residual neural retina is invaded by heavily pigmented cells. CONCLUSIONS: The initial ocular target of the mivit gene is the RPE, which is abnormal for many weeks before photoreceptor cells differentiate and become demonstrably affected. The authors hypothesize that the slowly progressive photoreceptor cell degeneration is secondary to abnormal function of the RPE. This mutation serves to refocus attention on critical influences of the RPE on function and maintenance of photoreceptor cells.

Increased cell genesis in retinal pigment epithelium of perinatal vitiligo mutant mice.

PURPOSE: To compare cell proliferation in vitiligo and control mouse retinal pigment epithelium (RPE) perinatally. METHODS: C57BL/6J-mivit/mivit mice and congenic +/+ controls were injected once with bromodeoxyuridine 1 hour before they were killed between embryonic day 18 and postnatal day 8. Wholemounts of Carnoy-fixed posterior eyecups, minus lens and neural retina, were stained immunohistochemically to detect DNA synthesis (bromodeoxyuridine incorporation) and mitotic cells (R3 antibody binding). Cells were counted in carefully controlled sampling sites, and total RPE area and face-view cell areas were calculated. Retinal pigment epithelial cell heights were measured on light and electron micrographs. RESULTS: Total surface areas of the mutant and control RPE monolayer were similar (I.E., RPE wholemount area was normal), but cell number was approximately doubled in the mutant RPE. By postnatal day 6, mutant cells had approximately 70% the face-view area as controls, but their heights were increased approximately 80%, so that cell volumes were near normal despite the higher packing density. Regional differences in cell size in the control RPE were absent in the mutant specimens. The mutant RPE showed an increased bromodeoxyuridine labeling index, as well as an absolute increase in the number of cells engaged in DNA synthesis and in mitosis. CONCLUSIONS: Cell genesis in the vitiligo RPE is quantitatively abnormal perinatally, well before the neural retina has been recognized to display functional or morphologic defects. Cells are being generated at an abnormally high rate, so that twice the normal number of cells are packed into a RPE of normal total area.