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http://www.sciencedaily.com/releases/2010/03/100321182915.htm
Science News Share Blog Cite Print Email BookmarkProof in Humans of
RNA Interference Using Targeted Nanoparticles
ScienceDaily (Mar. 23, 2010) — A California Institute of Technology (Caltech)-led
team of researchers and clinicians has published the first proof that a
targeted nanoparticle -- used as an experimental therapeutic and
injected directly into a patient's bloodstream -- can traffic into
tumors, deliver double-stranded small interfering RNAs (siRNAs), and
turn off an important cancer gene using a mechanism known as RNA
interference (RNAi).
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Moreover, the team provided the first demonstration that this new type
of therapy, infused into the bloodstream, can make its way to human
tumors in a dose-dependent fashion -- i.e., a higher number of
nanoparticles sent into the body leads to a higher number of
nanoparticles in the tumor cells.
These results, published in the March 21 advance online edition of the
journal Nature, demonstrate the feasibility of using both nanoparticles
and RNAi-based therapeutics in patients, and open the door for future "game-changing"
therapeutics that attack cancer and other diseases at the genetic level,
says Mark Davis, the Warren and Katharine Schlinger Professor of
Chemical Engineering at Caltech, and the research team's leader.
The discovery of RNA interference, the mechanism by which double strands
of RNA silence genes, won researchers Andrew Fire and Craig Mello the
2006 Nobel Prize in Physiology or Medicine. The scientists first
reported finding this novel mechanism in worms in a 1998 Nature paper.
Since then, the potential for this type of gene inhibition to lead to
new therapies for diseases like cancer has been highly touted.
"RNAi is a new way to stop the production of proteins," says Davis. What
makes it such a potentially powerful tool, he adds, is the fact that its
target is not a protein. The vulnerable areas of a protein may be hidden
within its three-dimensional folds, making it difficult for many
therapeutics to reach them. In contrast, RNA interference targets the
messenger RNA (mRNA) that encodes the information needed to make a
protein in the first place.
"In principle," says Davis, "that means every protein now is druggable
because its inhibition is accomplished by destroying the mRNA. And we
can go after mRNAs in a very designed way given all the genomic data
that are and will become available."
Still, there have been numerous potential roadblocks to the application
of RNAi technology as therapy in humans. One of the most problematic has
been finding a way to ferry the therapeutics, which are made up of
fragile siRNAs, into tumor cells after direct injection into the
bloodstream. Davis, however, had a solution. Even before the discovery
of RNAi, he and his team had begun working on ways to deliver nucleic
acids into cells via systemic administration. They eventually created a
four-component system -- featuring a unique polymer -- that can self-assemble
into a targeted, siRNA-containing nanoparticle. The siRNA delivery
system is under clinical development by Calando Pharmaceuticals, Inc., a
Pasadena-based nanobiotech company.
"These nanoparticles are able to take the siRNAs to the targeted site
within the body," says Davis. Once they reach their target -- in this
case, the cancer cells within tumors -- the nanoparticles enter the
cells and release the siRNAs.
The scientific results described in the Nature paper are from a Phase I
clinical trial of these nanoparticles that began treating patients in
May 2008. Phase I trials are, by definition, safety trials; the idea is
to see if and at what level the drug or other therapy turns harmful or
toxic. These trials can also provide an in-human scientific proof of
concept -- which is exactly what is being reported in the Nature paper.
Using a new technique developed at Caltech, the team was able to detect
and image nanoparticles inside cells biopsied from the tumors of several
of the trial's participants. In addition, Davis and his colleagues were
able to show that the higher the nanoparticle dose administered to the
patient, the higher the number of particles found inside the tumor cells
-- the first example of this kind of dose-dependent response using
targeted nanoparticles.
Even better, Davis says, the evidence showed the siRNAs had done their
job. In the tumor cells analyzed by the researchers, the mRNA encoding
the cell-growth protein ribonucleotide reductase had been degraded. This
degradation, in turn, led to a loss of the protein.
More to the point, the mRNA fragments found were exactly the length and
sequence they should be if they'd been cleaved in the spot targeted by
the siRNA, notes Davis. "It's the first time anyone has found an RNA
fragment from a patient's cells showing the mRNA was cut at exactly the
right base via the RNAi mechanism," he says. "It proves that the RNAi
mechanism can happen using siRNA in a human."
"There are many cancer targets that can be efficiently blocked in the
laboratory using siRNA, but blocking them in the clinic has been elusive,"
says Antoni Ribas, associate professor of medicine and surgery at UCLA's
Jonsson Comprehensive Cancer Center. "This is because many of these
targets are not amenable to be blocked by traditionally designed anti-cancer
drugs. This research provides the first evidence that what works in the
lab could help patients in the future by the specific delivery of siRNA
using targeted nanoparticles. We can start thinking about targeting the
untargetable."
"Although these data are very early and more research is needed, this is
a promising study of a novel cancer agent, and we are proud of our
contribution to the initial clinical development of siRNA for the
treatment of cancer," says Anthony Tolcher, director of clinical
research at South Texas Accelerated Research Therapeutics (START).
"Promising data from the clinical trials validates our years of research
at City of Hope into ribonucleotide reductase as a target for novel
gene-based therapies for cancer," adds coauthor Yun Yen, associate
director for translational research at City of Hope. "We are seeing for
the first time the utility of siRNA as a cancer therapy and how
nanotechnology can target cancer cells specifically."
The Phase I trial -- sponsored by Calando Pharmaceuticals -- is
proceeding at START and UCLA's Jonsson Comprehensive Cancer Center, and
the clinical results of the trial will be presented at a later time. "At
the very least, we've proven that the RNAi mechanism can be used in
humans for therapy and that the targeted delivery of siRNA allows for
systemic administration," Davis says. "It is a very exciting time."
In addition to Davis, Ribas, Tolcher, and Yen, the coauthors on the
Nature paper are Caltech graduate students Jonathan Zuckerman (an MD/PhD
student doing his MD work at UCLA) and Chung Hang Choi; former Caltech
graduate student Christopher Alabi, now a postdoctoral scholar at the
Massachusetts Institute of Technology; David Seligson, director of the
UCLA Tissue Array Core Facility at the David Geffen School of Medicine;
and Jeremy Heidel, who is currently a consultant for Calando
Pharmaceuticals.
The work described in the paper was supported in part by the National
Cancer Institute and the Daljit S. and Elaine Sarkaria Biomarker
Laboratories. Caltech, Davis, and Heidel have a financial interest in
Calando Pharmaceuticals.-
2010 NAF Annual Membership Meeting Presentations.
http://ataxia.org/events/Presentations-Chicago2010.aspx
Power point presentation from the 53rd NAF annual membership meeting -
Grazia Isaya, MD, PhD: Friedreich Ataxia Research and Prospects for
Therapy.
http://www.ataxia.org/pdf/2010_Presentations/Friedreichs_Ataxia_Research(Isaya).pdf
http://www.mdvu.org/emove/article.asp?ID=1248
Subject: Riluzole for Cerebellar Ataxia
Date: 3/19/2010
Riluzole improves symptoms of cerebellar ataxia, according to a new
placebo-controlled study. Riluzole is an approved treatment for ALS that
may reduce hyperexcitability of neurons in the deep cerebellar nuclei.
Forty patients with cerebellar ataxia of different etiologies (including
hereditary ataxias, MSA-C, FXTAS, and Friedreich’s ataxia) were
randomized to placebo or 50 mg riluzole twice daily for 8 weeks.
Baseline scores on the International Cooperative Ataxia Rating Scale
ranged from 12 to 70 out of 100.
The number of patients with at least a 5-point improvement in the ICARS
was greater in the riluzole group than in the placebo group at both 4
weeks (9/19 vs. 1/19, odds ratio=16.2) and 8 weeks (13/19 vs. 1/19,
OR=39). Riluzole improved the mean ICARS score by 7 points versus a 0.2
point worsening for placebo. Improvements were seen on the subscores for
static function, kinetic function, and dysarthria, but not for
oculomotor function. One patient experienced a slight increase in
alanine aminotransferase.
The results, the authors conclude, “may warrant the long-term use of
riluzole in chronic cerebellar ataxia.”
Riluzole in cerebellar ataxia: A randomized, placebo-controlled pilot
trial
G Ristori, S Romano, A Visconti, S Cannoni, M Spadaro, M Frontali, FE
Pontieri, N Vanacore, M Salvetti
Neurology 2010;74:839-845
E-MOVE Editor: Richard Robinson, NASW, WE MOVE
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9T-4YP16SS-2&_user=10&_coverDate=03%2F24%2F2010&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view
=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=
10&md5=646e51532099135d6ca92e908f7b92a3
Friedreich Ataxia Scientific News
Ataxia Friedreich, Friedreich's Ataxia, Frataxina, Frataxin,
Neurodegeneración, Neurodegeneration.
Friday, March 26, 2010
Friedreich ataxia presenting as sudden cardiac death in childhood:
Clinical, genetic and pathological correlation, with implications for
genetic testing and counselling
Neuromuscular Disorders
Article in Press, Corrected Proof, doi:10.1016/j.nmd.2010.02.019
Nada Querciaa, Gino R. Somersb, William Hallidayb, Paul F. Kantorc,
Brenda Banwelld and Grace Yoona, d,
a Division of Clinical and Metabolic Genetics, Hospital for Sick
Children, University of Toronto, Toronto, Canada
b Department of Paediatric Laboratory Medicine, Hospital for Sick
Children, University of Toronto, Toronto, Canada
c Department of Paediatrics, Division of Cardiology, Hospital for Sick
Children, Canada
d Department of Paediatrics, Division of Neurology, Hospital for Sick
Children, University of Toronto, Toronto, Canada
Received 5 November 2009; revised 22 January 2010; accepted 23 February
2010. Available online 24 March 2010.
Abstract
Friedreich ataxia (FRDA) is the most common cause of childhood onset
ataxia. We report on a 4 year old boy who suffered sudden cardiac death
and was found to have a dilated cardiomyopathy with left ventricular
hypertrophy on post-mortem studies. Molecular genetic testing
subsequently confirmed the diagnosis of Friedreich ataxia. To our
knowledge, this is the first report of Friedreich ataxia presenting as
sudden cardiac death in early childhood.
http://www.sciencedaily.com/releases/2010/03/100325143101.htm
New Period of Brain 'Plasticity' Created With Transplanted Embryonic
Cells
ScienceDaily (Mar. 25, 2010) — UCSF scientists report that they were
able to prompt a new period of "plasticity," or capacity for change, in
the neural circuitry of the visual cortex of juvenile mice. The approach,
they say, might some day be used to create new periods of plasticity in
the human brain that would allow for the repair of neural circuits
following injury or disease.
The strategy -- which involved transplanting a specific type of immature
neuron from embryonic mice into the visual cortex of young mice -- could
be used to treat neural circuits disrupted in abnormal fetal or
postnatal development, stroke, traumatic brain injury, psychiatric
illness and aging.
Like all regions of the brain, the visual cortex undergoes a highly
plastic period during early life. Cells respond strongly to visual
signals, which they relay in a rapid, directed way from one appropriate
cell to the next in a process known as synaptic transmission. The
chemical connections created in this process produce neural circuitry
that is crucial for the function of the visual system. In mice, this
critical period of plasticity occurs around the end of the fourth week
of life.
The catalyst for the so-called critical period plasticity in the visual
cortex is the development of synaptic signaling by neurons that release
the inhibitory neurotransmitter GABA. These neurons receive excitatory
signals from other neurons, thus helping to maintain the balance of
excitation and inhibition in the visual system.
In their study, published in the journal Science, (Vol. 327. no. 5969,
2010), the scientists wanted to see if the embryonic neurons, once they
had matured into GABA-producing inhibitory neurons, could induce
plasticity in mice after the normal critical period had closed.
The team first dissected the immature neurons from their origin in the
embryonic medial ganglionic eminence (MGE) of the embryonic mice. Then
they transplanted the MGE cells into the animals' visual cortex at two
different juvenile stages. The cells, targeted to the visual cortex,
dispersed through the region, matured into GABAergic inhibitory neurons,
and made widespread synaptic connections with excitatory neurons.
The scientists then carried out a process known as monocular visual
deprivation, in which they blocked the visual signals to one eye in each
of the animals for four days. When this process is carried out during
the critical period, cells in the visual cortex quickly become less
responsive to the eye deprived of sensory input, and become more
responsive to the non-deprived eye, creating alterations in the neural
circuitry. This phenomenon, known as ocular dominance plasticity,
greatly diminishes as the brain matures past this critical postnatal
developmental period.
The team wanted to see if the transplanted cells would affect the visual
system's response to the visual deprivation after the critical period.
They studied the cells' effects after allowing them to mature for
varying lengths of time. When the cells were as young as 17 days old or
as old as 43 days old, they had little impact on the neural circuitry of
the region. However, when they were 33-39 days old, their impact was
significant. During that time, monocular visual deprivation shifted the
neural responses away from the deprived eye and toward the non-deprived
eye, revealing the state of ocular dominance plasticity.
Naturally occurring, or endogenous, inhibitory neurons are also around
33-39 days old when the normal critical period for plasticity occurs.
Thus, the transplanted cells' impact occurred once they had reached the
cellular age of inhibitory neurons during the normal critical period.
The finding, the team says, suggests that the normal critical period of
plasticity in the visual cortex is regulated by a developmental program
intrinsic to inhibitory neurons, and that embryonic inhibitory neuron
precursors can retain and execute this program when transplanted into
the postnatal cortex, thereby creating a new period of plasticity.
"The findings suggest it ultimately might be possible to use inhibitory
neuron transplantation, or some factor that is produced by inhibitory
neurons, to create a new period of plasticity of limited duration for
repairing damaged brains," says author Sunil P. Gandhi, PhD, a
postdoctoral fellow in the lab of Michael Stryker, PhD, professor of
physiology and a member of the Keck Center for Integrative Neurosciences
at UCSF. "It will be important to determine whether transplantation is
equally effective in older animals."
Likewise, "the results raise a fundamental question: how do these cells,
as they pass through a specific stage in their development, create these
windows of plasticity?" says author Derek G. Southwell, PhD, a student
in the lab of Arturo Alvarez-Buylla, PhD, Heather and Melanie Muss
Professor of Neurological Surgery and a member of the Eli and Edythe
Broad Center of Regeneration Medicine and Stem Cell Research at UCSF.
The findings could be relevant to understanding why learning certain
behaviors, such as language, occurs with ease in young children but not
in adults, says Alvarez-Buylla. "Grafted MGE cells may some day provide
a way to induce cortical plasticity and learning later in life."
The findings also complement two other recent UCSF studies using MGE
cells to modify neural circuits. In a collaborative study among the
laboratories of Scott Baraban, PhD, professor of neurological surgery;
John Rubenstein, MD, PhD, professor of psychiatry, and Alvarez-Buylla,
the cells were grafted into the neocortex of juvenile rodents, where
they reduced the intensity and frequency of epileptic seizures. (Proceedings
of the National Academy of Science, vol. 106, no. 36, 2009). Other teams
are exploring this tactic, as well.
In the other study (Cell Stem Cell, vol. 6, issue 3, 2010), UCSF
scientists reported the first use of MGEs to treat motor symptoms in
mice with a condition designed to mimick Parkinson's disease. The
finding was reported by the lab of Arnold Kriegstein, MD, PhD, UCSF
professor of neurology and director of the Eli and Edythe Broad Center
of Regeneration Medicine and Stem Cell Research at UCSF, in
collaboration with Alvarez-Buylla and Krys Bankiewicz, MD, PhD, UCSF
professor of neurological surgery.
The other co-author of the plasticity study was Robert C. Froemke, PhD,
a postdoctoral fellow in the lab of Christoph Schreiner, MD, PhD,
professor and vice chair of otolaryngology.
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T1Y-4YN5PD1-1&_user=10&_coverDate=03%2F20%2F2010&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=
0&_userid=10&md5=771c46682c68955aee5b4e363a54c96c
Evidence that yeast frataxin is not an iron storage protein In Vivo
Alexandra Seguina, 1, Robert Sutaka, 1, Anne-Laure-Bulteaub, Richard
Garcia-Serresc, Jean-Louis Oddouc, Sophie Lefevrea, Renata Santosa,
Andrew Dancisd, Jean-Michel Camadroa, Jean-Marc Latourc, 2 and Emmanuel
Lesuissea, 2, ,
a Laboratoire Mitochondries, Métaux et Stress oxydant, Institut Jacques
Monod, CNRS-Université Paris Diderot, France.
b Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement,
Université Paris 7, Paris, France.
c CEA, iRTSV, LCBM, 38054 Grenoble Cedex 9, France; CNRS, UMR5249,
Grenoble, France; Université Joseph Fourier, 38054 Grenoble Cedex 9,
France
d University of Pennsylvania, Department of Medicine, Division of
Hematology/Oncology, BRBII Room 731, 431 Curie Blvd, Philadelphia PA
19104, USA
Received 10 October 2009; revised 14 March 2010; accepted 16 March 2010.
Available online 20 March 2010.
Abstract
Yeast cells deficient in the yeast frataxin homolog (Yfh1p) accumulate
iron in their mitochondria. Whether this iron is toxic, however, remains
unclear. We showed that large excesses of iron in the growth medium did
not inhibit growth and did not decrease cell viability. Increasing the
ratio of mitochondrial iron-to-Yfh1p by decreasing the steady-state
level of Yfh1p to less than 100 molecules per cell had very few
deleterious effects on cell physiology, even though the mitochondrial
iron concentration greatly exceeded the iron-binding capacity of Yfh1p
in these conditions. Mössbauer spectroscopy and FPLC analyses of whole
mitochondria or of isolated mitochondrial matrices showed that the
chemical and biochemical forms of the accumulated iron in mitochondria
of mutant yeast strains (∆yfh1, ∆ggc1, ∆ssq1) displayed a nearly
identical distribution. This was also the case for ∆ggc1 cells, in which
Yfh1p was overproduced. In these mitochondria, most of the iron was
insoluble, and the ratio of soluble-to-insoluble iron did not change
when the amount of Yfh1p was increased up to 4,500 molecules per cell.
Our results do not privilege the hypothesis of Yfh1p being an iron
storage protein in vivo.
Keywords: Yeast frataxin; iron; oxidative stress; yfh1; ggc1; Mössbauer;
mitochondria
Corresponding author. Institut Jacques Monod, CNRS-Université Paris
Diderot, Bâtiment Buffon, 15 rue Hélène Brion, 75205 Paris cedex 13,
France. Tel.: +33 157278028; fax: +33157278101.
http://www.springerlink.com/content/mnt01q7828063ghr/
Pathology and pathogenesis of sensory neuropathy in Friedreich’s ataxia
Jennifer A. Morral1, Ashley N. Davis1, Jiang Qian2, Benjamin B. Gelman3
and Arnulf H. Koeppen1, 2, 4
(1) Research Service (151), VA Medical Center, 113 Holland Ave, Albany,
NY 12208, USA
(2) Department of Pathology and Laboratory Medicine, Albany Medical
College, Albany, NY 12208, USA
(3) Department of Pathology and Laboratory Medicine, University of Texas
Medical Branch, Galveston, TX 77555, USA
(4) Department of Neurology, Albany Medical College, Albany, NY 12208,
USA
Received: 17 December 2009 Revised: 17 March 2010 Accepted: 17 March
2010 Published online: 26 March 2010
Abstract
Friedreich’s ataxia (FRDA) causes a complex neuropathological phenotype
with characteristic lesions of dorsal root ganglia (DRG); dorsal spinal
roots; dorsal nuclei of Clarke; spinocerebellar and corticospinal tracts;
dentate nuclei; and sensory nerves. This report presents a systematic
morphological analysis of sural nerves obtained by autopsy of six
patients with genetically confirmed FRDA. The outstanding lesion
consisted of lack of myelinated fibers whereas axons were present in
normal numbers. On cross-sections, only 11% of all class III-β-tubulin-positive
axons were myelinated in FRDA, contrasting with 36% in normal control
nerves. Despite their paucity, thin myelinated fibers assembled compact
sheaths containing the peripheral myelin proteins PMP-22, P0, and myelin
basic protein. The nerves displayed major modifications in Schwann cells
that were apparent by laminin 2 and S100α immunocytochemistry. Few
S100α-immunoreactive cells remained detectable whereas laminin 2
reaction product was abundant. The normal honeycomb-like distribution of
laminin 2 around myelinated fibers was replaced by confluent regions of
reaction product that enveloped clusters of closely apposed thin axons.
Electron microscopy not only confirmed the lack of myelin but also
showed abnormal Schwann cells and axons. Ferritin localized to normal
Schwann cell cytoplasm. In the sensory nerves of patients with FRDA, the
distribution of this protein strongly resembled laminin 2, but there was
no net increase of the total ferritin-reactive area. Ferroportin
reaction product occurred in all axons of sural nerves in FRDA, which
was at variance with dorsal spinal roots. In the pathogenesis of sensory
neuropathy in FRDA, two mechanisms are likely: hypomyelination due to
faulty interaction between axons and Schwann cells; and slow axonal
degeneration. Neurons of DRG, satellite cells, Schwann cells, and axons
of sensory nerves and dorsal spinal roots derive from the neural crest,
and hypomyelination in FRDA may be attributed to defects of regulation
or migration of shared precursor cells. Sural nerves in FRDA showed no
convincing change in ferritin and ferroportin, militating against local
iron dysmetabolism. The result stands out in contrast to the previously
reported changes in dorsal spinal roots of patients with FRDA.
Keywords Axons - Friedreich’s ataxia - Laminin - Myelin sheath - Neural
crest - S100 protein - Schwann cells - Sural nerve
Arnulf H. Koeppen
Email: arnulf.koeppen@med.va.gov
Hum Mol Genet. 2010 Feb 25.
http://www.ncbi.nlm.nih.gov/pubmed/20154340?dopt=Abstract
Efficient recovery of dysferlin deficiency by dual adeno-associated
vector-mediated gene transfer.
Lostal W, Bartoli M, Bourg N, Roudaut C, Bentaïb A, Miyake K, Guerchet
N, Fougerousse F, McNeil P, Richard I.
Généthon, CNRS UMR8587 LAMBE, 1, rue de l'Internationale, 91000 Evry,
France and.
Deficiency of the dysferlin protein presents as two major clinical
phenotypes: limb-girdle muscular dystrophy type 2B and Miyoshi myopathy.
Dysferlin is known to participate in membrane repair, providing a
potential hypothesis to the underlying pathophysiology of these diseases.
The size of the dysferlin cDNA prevents its direct incorporation into an
adeno-associated virus (AAV) vector for therapeutic gene transfer into
muscle. To bypass this limitation, we split the dysferlin cDNA at the
exon 28/29 junction and cloned it into two independent AAV vectors
carrying the appropriate splicing sequences. Intramuscular injection of
the corresponding vectors into a dysferlin-deficient mouse model led to
the expression of full-length dysferlin for at least 1 year. Importantly,
systemic injection in the tail vein of the two vectors led to a
widespread although weak expression of the full-length protein.
Injections were associated with an improvement of the histological
aspect of the muscle, a reduction in the number of necrotic fibers,
restoration of membrane repair capacity and a global improvement in
locomotor activity. Altogether, these data support the use of such a
strategy for the treatment of dysferlin deficiency.
PMID: 20154340 [PubMed - as supplied by publisher]
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