researchA PublicAtion of The ReseaRch InsTITuTe aT naTIonwIde chIldRen’s hospITal

Genetics of Heart Defects

feature Stories• Genetics of Heart Defects

• Classifying Concussions

• Disarming Killer T Cells

• Bypassing the Brain’s Barrier

Might a Defective Signaling Pathway Lead to Left-Sided Heart Malformations?

features

4 Genetics of Heart Defects Molecular proof that three distinct cardiac defects share an origin

8 Classifying Concussions Risk factors help predict how concussed kids will recover

12 Tracking the Mysterious Death of Killer T Cells Could a mechanism for silencing immunity improve the outcome of viral

gene therapy?

14 Bypassing the Brain’s Barrier A new strategy evades a biological obstacle to gene therapy success

extras

7 A Home to National Clinical Trials Nationwide Children’s Hospital has been selected to join two new clinical

research trial networks, a designation bestowed only to preeminent

research facilities in the nation

11 Research on the Road New mobile research lab targets hard-to-reach participants

16 Technology in Practice A risk-assessment technology developed at Nationwide Children’s is

helping physicians and patients communicate more effectively

ON THe COveR Kim L. McBride, MD, examines Notch protein distribution throughout the interior and on the surface of a cell. New research shows that changes in Notch signaling could promote heart defects.

leadeRshIp The Research Institute at nationwide children’s hospital

John a. Barnard, MdPresident

lauren o. Bakaletz, phdVice President, Basic Sciences Research

Kelly Kelleher, Md, MphVice President, Health Services Research

william e. smoyer, MdVice President, Clinical and Translational Research

Grant Morrow III, MdMedical Director

daniel R. MannVice President, Research Administration and Operations

Katherine s. MilemPresident, Research Business Services

researchWriter and editorMelissa hamilton

Art DirectorTanya Bender

PhotographersBrad smithdan smith

Manager, Research CommunicationsJan arthur

Contact us at ResearchMagazine@NationwideChildrens.org

research is dedicated to the

mission of informing and inspiring

readers by highlighting scientific

performance at The Research Institute

at Nationwide Children’s Hospital.

This publication is produced

biannually by the Marketing and

Public Relations Department at

Nationwide Children’s Hospital.

Table of Contents

research | 3

sPRING/suMMeR 2009

14 Injection of a gene therapy virus,

AAV9, successfully bypassed the blood

brain barrier and targeted neurons,

the carriers of electrical signals in the

nervous system, and astrocytes, the

most abundant cells in the nervous

system. Neurons that were successfully

infected by AAV9 are shown in yellow;

infected astrocytes are teal.

The finding that AAV9 efficiently enters

the nervous system after injection into

the bloodstream provides potential for

new therapies and could lead to better

understanding of neurological disease.

research | 54 | research

THe MysTeRy Of LefT-HeART MAlforMAtionS Congenital cardiovascular malformations are among the most common birth defects in the United States. One group of heart defects known as left ventricular outflow tract obstruction (LVOTO) malformations arise from obstruction of blood flow through the developing left ventricle and aorta. These malformations include aortic valve stenosis, coarctation of the aorta and hypoplastic left heart syndrome. Without major surgery during

the first few days of life, many newborns with an LVOT malformation, especially those with hypoplastic left heart syndrome, will die.

Presumably, these defects arise when the inner lining of the heart or aorta develops improperly, but the basic, disease-causing mechanisms remain unclear. Scientists believe they are likely a complex mix of defects in one or more genes, chromosomal abnormalities and possibly environmental exposures.

Despite the myriad of mutations that could be responsible for each of the LVOT malformations, faculty members at The Research Institute are discovering that the most telling explanation of disease devel-opment could be revealed by studying the disorders together as a group.

“Clinicians traditionally thought of these heart defects as separate entities,” said Kim L. McBride, MD, principal investigator in the Center for Molecular and Human Genetics at The Research Institute. “More recent views have grouped them together based on findings from cardiac developmental biology, observations of clustering in families, and our own previous work on inheritance analysis.”

LINKeD By A MOLeCuLAR MeCHANIsM Research conducted by Dr. McBride and colleagues reveals, for the first time, a common molecular mechanism for aortic

Genetics of Heart DefectsPROOf THAT THRee DIsTINCT CARDIAC DefeCTs sHARe AN ORIGIN

Like a windstorm that leaves one household without power, another with downed trees and a third with roof damage, scientists are finding that congenital heart conditions can have differing effects, yet stem from a common biological event.

valve stenosis, coarctation of the aorta and hypoplastic left heart syndrome. Although all three conditions involve the left side of the heart, a molecular similarity between the three defects had not yet been identified.

During the study, which appears in Human Molecular Genetics, 91 patients with LVOT malformations had their DNA screened for mutations in NOTCH1, a gene that has been linked to an aortic valve condition and that is part of a signaling pathway involved in multiple developmental processes. These patients’ DNA was then compared to that of 216 people without LVOT malformations.

Results showed that NOTCH1 gene mutations are present in some individuals with aortic valve stenosis, coarctation of the aorta and hypoplastic left heart syndrome. With the help of Dr. Susan Cole, assistant professor in the College of Biological Sciences at The Ohio State University, several of these mutations were shown to affect Notch pathway signaling in the cell. This suggests that Notch signaling plays a vital role during cardiovascular development and relatively minor changes may promote LVOT defects. “These findings support our previous idea that these defects may share a common developmental pathogenesis,” said Dr. McBride. “They also emphasize the hereditary nature of these defects and explain why one can find individuals in the same family with different forms of LVOT malformations.”

seARCHING fOR A GeNeTIC LINKSince Dr. McBride’s group has established that there is a strong and complex genetic component to LVOT malformations, their focus is now to pinpoint the genes behind the disorders. Using a variety of study designs for gene mapping, the team is analyzing the pattern of inheritance of selected genes in signaling pathways important in cardiac development.

Most recently, they conducted the first-ever study to “gene hunt” among families with multiple LVOT malformations, grouping the malformations together as though they were one disorder. “Grouping the individual malformations together as one disease allowed us to greatly increase the ability to map the locations of the responsible genes,” said Dr. McBride.

Families with at least two members with an LVOT malformation were included in the study and each underwent echocardiography to assess for any structural heart abnormalities. After analyzing DNA markers from these families, researchers found evidence for linkage to three chromosomes for the combined LVOT malformations and for the disorders individually.

These data provide the initial phase for gene identification of these LVOT malformations, a basic-science discovery that could be translated into improved patient care. “Identifying a specific gene also now holds hope for eventually providing better risk counseling for families,” said Dr. McBride.

Knowing that LVOT malformations share a common molecular mechanism is a big step forward… – DR. KIM L. MCBRIDe

CONsequeNCes Of A TRAPPeD PROTeIN How could minor changes in the Notch protein sequence promote heart defects? Recent research has shown that some mutations in the NOTCH1 gene affect where the protein appears in the cell, trapping it and making it non-functional.

1) Notch1 protein distribution (green) as it appears normally, throughout the cell interior and on the cell surface.

2) Distribution of mutant Notch1 protein (green) trapped near the cell’s nucleus (blue).

2)1)

research | 76 | research

Nationwide Children’s

has been selected to

join two new clinical

research trial networks, one

through the Muscular Dystrophy

Association and the other through

the Cystic Fibrosis Foundation.

This designation is bestowed only

to preeminent research facilities in

the nation. Clinical trial networks

bring together clinical research

centers throughout the nation to

centralize and standardize the

research process.

Faculty members at Nationwide

Children’s also serve on more than

40 study sections of national review

panels across a full range of pediatric

health topics. Section members are

considered experts in their field and

assess the scientific and technical

merit of research conducted by

their peers.

Gastrointestinal cell and Molecular Biology

host Interactions with Bacterial pathogens

pediatric heart network

Blood cancers

skeletal Muscle Biology and

exercise physiology

neural development, plasticity and Regeneration They’re

Consulting Our Experts

Faculty members at nationwide children’s serve on more than 40 study sections of national review panels across a full range of pediatric health topics.

Web exclusive To view a full list of national review panels that include experts from Nationwide Children’s Hospital, visit us online at www.NationwideChildrens.org − keyword search: panels.

sCIeNTIfIC TRANsLATION THROuGH CLINICAL collAborAtion In order to progress toward gene discovery, Dr. McBride’s team must maintain the clinical collaborations that have been so vital in their present research. While Dr. McBride’s team utilizes animal models during their studies, no existing animal models precisely mimic LVOT malformations. This is why much of their research directly involves real-life patient samples.

The complex nature of this translational work necessitates close collaborations with a variety of skilled people including a genetic counselor who recruits patients and families, obtains detailed medical and family histories and collects blood for lab studies. This information then becomes part of an extensive clinical database that serves as a repository for scientific study.

Dr. McBride also collaborates with clinical faculty at Nationwide Children’s including surgeons who provide heart tissue samples donated by patients during cardiac surgery. “It would be impossible to conduct this research without the teamwork of our on-site surgeons because the tissue samples are too fragile to be shipped from one location to another,” said Dr. McBride. “It also helps that we are able to collaborate with doctors who are leaders in the treatment of hypoplastic left heart syndrome.”

Ultimately, the genetic information obtained by Dr. McBride and his colleagues may result in genetic tests to identify at-risk pregnancies early and help develop unique treatments and improved surgical techniques.

“Although we are at the beginning of our hunt for the causes of these devastating heart malformations, we now have a

better view of our target,” said Dr. McBride. “Knowing that LVOT malformations share a common molecular mechanism is a big step forward, as it allows us to more effectively focus our efforts, provides the first clues to the underlying pathogenesis, and hopefully will allow improved genetic counseling for affected families.”

Further reading: McBride KL, Riley MF, Zender GA, Fitzgerald-Butt SM, Towbin JA, Belmont JW, Cole SE. NOTCH1 mutations in individuals with left ventricular outflow tract malformations reduce ligand-induced signaling. Hum Mol Genet. 2008 Sep 15;17(18):2886-93.

McBride KL, Zender GA, Fitzgerald-Butt SM, Koehler D, Menesses-Diaz A, Fernbach S, Lee K, Towbin JA, Leal S, Belmont JW. Linkage analysis of left ventricular outflow tract malformations (aortic valve stenosis, coarctation of the aorta, and hyp-oplastic left heart syndrome). Eur J Hum Genet. 2009

Collaborative research between Kim L. McBride, MD, and Susan Cole, PhD, are revealing new information about how some heart defects develop.

research | 98 | research

A well-known, albeit unfortunate story among Dr. Keith Yeates’ family is that he toppled down the stairs in a mobile baby walker during childhood. Although he was not evaluated for head trauma, the fall left him with two prominent black eyes, which Dr. Yeates assumes were the result of a basilar skull fracture.

The clinical neuropsychologist and director of the Center for Biobehavioral Health at The Research Institute cites this childhood tale as a personal example of how head trauma and its

potentially long-lasting effects are not so easily identified.

Fortunately, his almost 20 years of clinical and research experience at Nationwide Children’s Hospital is helping to reverse this reality. By studying brain development and outcomes of brain injury, Dr. Yeates and his team are working to help physicians and families understand risk factors associated with traumatic brain injury and their expected outcomes.

In relation to concussions, one of the most common childhood head injuries, researchers are learning that multiple physical and psychosocial factors play a role in how children will recover.

CHARACTeRIsTICs Of CONCussION A concussion is a mild injury to the brain caused by a blow to the head. It’s estimated that more than a half million kids in the United States go to the hospital each year with this injury, an average of a kid per minute, every minute of every day.

Although these mild brain injuries are common among children, Dr. Yeates says that not all concussions should be treated equally. “There is often an assumption that all mild head injuries are the same, that all concussions are the same. But, conventional definitions of concussion and mild head injury actually encompass a wide range of severity,” he said.

This wide range of severity can range from injuries that lead to a 20-minute loss of consciousness and obvious lesions on brain imaging, to those that only involve feeling foggy, without being unconscious, and no findings on neuroimaging. Yet the level of severity is not the only determinant of patient outcome. “It’s clear clinically that some kids with brain injuries do surprisingly well and others do very poorly,” said Dr. Yeates. “Although there is a relationship between how severe an injury is and how kids do, there is a lot of variability,” he said.

WHeN CONCussION syMPTOMs LINGeR In an effort to clarify risk factors related to concussion outcomes, Dr. Yeates and colleagues recently examined nearly 200 children with concussion and followed them for a year. The study, which appears in Pediatrics, revealed that while most kids had few prob-lems, one out of every four experienced significant post-concussive symptoms, some of which never fully resolved. “There is a group of kids that are at risk and do seem to be able to continue to have these symptoms even up to a year after their injury,” said Dr. Yeates.

Still, he says this study is not cause for alarm. In reality, it provides reassurance for parents of kids who suffer first-time concussions because more often than not children recover fully within a short amount of time. Despite this, the study also shows that kids who are at risk because their concussions are more severe need to be monitored for a longer period of time as their symptoms may last longer.

However, physicians aren’t always able to predict who will have long-term issues. While symptoms like headaches, fatigue and irritability can all be signs that a concussion is lingering, there is no test to show when a child has fully recovered.

Dr. Yeates believes classifying concussions as high-risk or low-risk may help physicians determine which patients need special atten-tion. “We’re trying to understand the factors that relate to recovery.”

INjuRy seveRITy PLus fAMILy DyNAMICs As previously indicated, one recovery-related factor seems to be severity of injury. Results from the aforementioned study showed that children whose concussions resulted in a loss of consciousness, disorientation or post-traumatic amnesia, or an abnormal MRI were more likely to have symptoms that persisted.

Yet, additional research suggests there might be more at work. “Given the amount of time kids spend with their parents and in the family setting, it seems possible that these are factors in recovery,” said Dr. Yeates. “Is it the injury that accounts for post-concussive symptoms? Or is it other factors like how the family copes or how the child reacts? What we’re finding is that it’s both.”

One of Dr. Yeates’ studies examined how family burden and parent distress following a child’s mild traumatic brain injury related to post-concussive symptoms. Overall, the results were encouraging, suggesting that family burden and parental distress following mild traumatic brain injury is typically limited. How-ever, data suggested that children’s post-concussive symptoms shortly after a minor injury may be a more powerful source of post-injury family burden and distress than the injury itself.

In conjunction with the University of Cincinnati, Dr. Yeates’ team is examining a potential intervention that uses a web-based tool to teach families and children with severe traumatic brain injury better problem-solving and communication skills as a way of reducing the parents’ distress and indirectly improving the children’s outcomes. “Our research suggests that it is important to do family-centered care, not just because it sounds like it is the nice thing to do, but because it actually makes a difference in how kids recover,” he said.

Classifying ConcussionsRIsK fACTORs HeLP PReDICT HOW CONCusseD KIDs WILL ReCOveR

Keith O. Yeates, PhD

Medical imaging techniques such as MRI help clinicians determine the severity of injury by revealing brain abnormalities related to trauma. Injury severity is one of the many recovery-related factors that Dr. Yeates’ team is examining in order to help classify concussions as high- or low-risk.

THe effeCTs Of BRAIN AGeResearch has also revealed that age is an important factor related to recovery. When it comes to brain injury, kids shouldn’t be compared to adults.

Consider this: Several studies that have focused specifically on adults with mild traumatic brain injury indicate that the presence of the APOE ε4 allele, a genetic risk factor, is associated with poorer neurobehavioral outcomes. However, after conducting a similar study in children with this allele, Dr. Yeates and col-leagues found that the APOE ε4 allele is not consistently related to the outcomes of mild traumatic brain injury in children.

Despite the fact that childhood brain injury needs to be evaluat-ed differently than similar injuries in adults, Dr. Yeates says there are more important age groups to compare. “There has been a lot of research in age-related differences in outcomes and the most significant difference isn’t between school-aged kids and adults. It is between preschool children and older children.”

Dr. Yeates says that in a more severe diffuse injury that tends to involve large regions of the brain, young kids actually don’t recover as quickly or as completely compared to older children. More research is needed to find out if the effects of mild traumatic brain injuries are also more pronounced for younger children.

The vulnerability of age is why it is important to investigate the effects that brain injuries have on aptitudes developed during childhood, such as social skills. An ongoing study in the Center for Biobehavioral Health is focused on social outcomes related to traumatic brain injury. “A lot of traumatic brain injury research has focused on kids’ cognitive skills, but the things that are most troublesome to kids and to their families have to do with how kids do behaviorally and socially,” said Dr. Yeates. “If you look at long-term predictions of how people turn out as adults, their social relationships as kids are better predictors of quality of life than school performance is.”

To aid in both cognitive and social development following brain injury, investigators in the Center for Biobehavioral Health, in conjunction with the University of Oregon, also are studying the outcomes of a school transition program that alerts schools when kids are coming back with traumatic brain injury, and providing the school with appropriate resources.

A MODeL Of BRAIN ReCOveRy The identification of risk factors that help to predict how patients will recover from brain injury, and a better understanding of how kids are affected post-injury, could change the way physicians and families deal with all-too-common concussions. The findings could also help inform models of recovery in other diseases that can affect the central nervous system, such as childhood cancer or prematurity.

Yet Dr. Yeates notes that as important prediction and aftercare are, methods of prevention should not be overlooked. “The biggest difference we could make in terms of disability and death for kids in the US would be doing a better job of preventing injury.”

Further reading: Yeates KO, Taylor HG, Rusin J, Bangert B, Dietrich A, Nuss K, Wright M, Nagin DS, Jones BL. Longitudinal trajectories of postconcussive symptoms in children with mild traumatic brain injuries and their relationship to acute clinical status. Pediatrics. 2009 Mar; 123 (3): 735-43.

Ganesalingam K, Yeates KO, Ginn MS, Taylor HG, Dietrich A, Nuss K, Wright M. Family burden and parental distress following mild traumatic brain injury in children and its relationship to post-concussive symptoms. J Pediatr Psychol. 2008 Jul;33(6):621-9.

Moran LM, Taylor HG, Ganesaligam K, Gastier-Foster JM, Frick J, Bangert B, Dietrich A, Nuss KE, Rusin J, Wright M, Yeates KO. Apolipoprotein E4 as a predictor of outcomes in pediatric mild traumatic brain injury. J Neurotrauma. 2009 Feb 18. [Epub ahead of print]

research | 1110 | research

Demographics are an important measure for ensuring that research findings are generalizable to large

populations. How can researchers increase a study’s demographic scope when it’s logistically difficult for a wide variety of people to participate?

They can go the distance.

In an effort to improve sampling and to reach target populations, Center for Biobehavioral Health Principal Investigator Brady Reynolds, PhD, has developed a “research lab on wheels.” This mobile lab is fully equipped to conduct studies off-site, in rural areas where participant recruitment is difficult.

The mobile lab is currently being used to extend Dr. Reynolds’ impulsivity research, which focuses on how specific impulsive behaviors are involved in an adolescent’s ability to stop or reduce smoking. Initial “on-the-road” data collection began in January 2009 in the Ohio Appalachia region.

Plans call for the mobile lab to be used for the next 10 to 15 years in studies by additional researchers, including research on parent safety practices and impulsivity and another focused on depression and teen smoking. “The opportunities for research are endless,” says Dr. Reynolds.

Research on the RoadNeW MOBILe ReseARCH LAB TARGeTs

HARD-TO-ReACH PARTICIPANTs

No Way to PlayKIDs ARe COMPeTING TOO sOON AfTeR CONCussIONs

A study from the Center for

Injury Research and Policy at

The Research Institute found that

41 percent of student athletes

across 100 US high schools who

suffered a concussion returned to

play too soon, against guidelines

set by the American Academy of

Neurology. In girl’s volleyball and

boy’s basketball and baseball,

more than half of the concussed

players returned to play before

fully recovered.

Web exclusive For “Concussion Tips and Symptoms: What Every Parent Should Know,” visit us online at www.NationwideChildrens.org − keyword search: concussion.

Inside the Mobile Lab

Every aspect of the mobile research lab was care-fully designed and strategically planned to utilize space and protect privacy during data collection.

PRIvATe BATHROOMSpace to collect urine samples to test for biomarkers. Such tests are more reliable than personal reports when measuring levels of cigarette smoking.

COMPuTeR CeNTeRA place for participants to complete specially-designed research tasks.

INTeRvIeW sPACe A private room used during one-on-one data collection.

OuTDOOR AWNING Provides shelter for conducting research or recruiting outside the lab.

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research | 1312 | research

What if your immune system could be switched on and off as needed to fight disease?

Although this scenario might seem suitable for a dramatic screenplay, in reality, scientists are edging closer to understanding how immunity is switched on and off during viral, bacterial and parasitic infection.

“Some of these pathogenic adversaries have learned to use these immune switches to gain an advantage during infection, said Christopher M. Walker, PhD, director of the Center for Vaccine and Immunity at The Research Institute. “The hepatitis C virus is an example. It can exploit one of the off switches to impair immunity in many infected people, resulting in life-long infection with the virus and greatly increased risk of serious liver diseases like cirrhosis and cancer.”

In an effort to improve the health of patients infected with the hepatitis C virus, Dr. Walker and his team are searching to “out-smart” this pathogen and switch immunity back on. As is often the case in research, this work has recently led Dr. Walker’s laboratory in an unexpected and important new direction, one that could have groundbreaking implications for gene therapy.

GeNe-CARRyING vIRuses Once considered futuristic, it is now possible to manipulate some simple viruses to deliver human genes. Through complex, technology-assisted processes, scientists remove the disease-causing genes from a simple virus and use the pathogen’s protective coating to package the functional human gene.

Known as viral gene therapy vectors, these manipulated viruses have been shown to cure genetic diseases in animals and are now being tested in humans. A common example is the adeno-associated virus (AAV), which has been converted to treat inherited diseases including cystic fibrosis, arthritis, muscular dystrophy and hemophilia.

Like an aircraft dropping needed supplies, scientists hope that vectors can be used to deliver the body’s missing genes. However, one potentially important barrier to this success could be the immune system. “Therapeutic proteins produced in diseased tissues differ from those inherited by the patient and so they may be viewed as foreign and threatening by the immune system,” said Dr. Walker.

Killer CD8 T cells are the most threatening component of the immune system. These white blood cells were designed by nature to destroy infected tissues and to interrupt production of new viruses. In gene therapy this normally beneficial response could lead to disaster.

A DesIRABLe fAILeD IMMuNe ResPONseYet, disaster may not be inevitable.

Remarkably, foreign proteins can be produced for a lifetime in mouse models without interference from the immune system. For many years it was thought that these therapeutic proteins were ignored by killer T cells, but recent studies by Dr. Walker’s team challenge this logic. Delivery of an AAV vector to leg muscles of mice did result in long-term production of the therapeutic protein, but instead of being ignored the protein engaged the immune system. Killer T cells even infiltrated the treated muscle, but failed in their mission to remove the muscle cells producing the therapeutic protein.

To Dr. Walker and his team, these findings

were reminiscent of studies in humans infected with the hepatitis C

virus. Dr. Walker’s team discovered that the killer T cells lost the ability to destroy tissues they

recognized as foreign and ultimately died, a process that has been observed in the liver of humans persistently infected with the hepatitis C virus.

A DeLICATe BALANCING ACT These studies indicate that the balance between success and failure in gene therapy is considerably more tenuous than first thought, relying on death of armed killer T cells. Unraveling mechanisms of killer T cell death may hold the key to treatment of diseases as diverse as chronic virus infection, in the case of hepatitis C, and success in gene therapy for inherited genetic diseases like muscular dystrophy or hemophilia B.

“It is surprising how two lines of research for diseases that appeared so dissimilar have converged in our laboratory,” said Walker. “Understanding the switches that lead to programmed death of T cells could at once allow us to restore immunity in a serious infectious disease like hepatitis C, and turn immunity off where it is not wanted in gene therapy.”

Moreover, this research serves as a reminder that current gene therapy strategies involve a delicate balancing act with host immunity. “As these findings indicate, cellular immune responses should also be considered as a potential risk to the success of gene transfer when using viral vectors,” said Dr. Walker.

Further reading: Velazquez VM, Bowen DG, Walker CM. Silencing of T lymphocytes by antigen-driven programmed death in recombinant adeno- associated virus vector-mediated gene therapy. Blood. 2009 Jan 15;113(3):538-45.

Tracking the Mysterious Death of Killer T CellsNeW ReseARCH Is sHOWING THAT GeNe THeRAPy DOes NOT evADe THe

IMMuNe sysTeM, As ORIGINALLy BeLIeveD. RATHeR, GeNe THeRAPy suCCess

ReLIes ON THe DeATH Of IMMuNe-sysTeM-ACTIvATeD KILLeR T CeLLs.

Illus

trat

ion

by A

ntho

ny W

eile

r

Christopher M. Walker, PhD

surprisingly, instead of ridding the cell of the foreign protein, killer T cells receive a mysterious death signal and begin to die. Their death leads to gene therapy success.

scientists deliver a “gene” to the leg muscle of a mouse model using a viral vector. In this case, the gene produces a protein called eGFp that flouresces green.

presence of the green fluorescence, which can be observed by the naked eye, confirms that the viral vector successfully delivered the gene and that the desired protein is being produced.

The immune system recognizes eGFp as foreign and killer T cells invade to destroy green muscle cells.

14 | research research | 15

One of the most important discoveries of brain biology was also the byproduct of a staining experiment gone awry.

During the late 1800s German bacteriologist Paul Ehrlich attempted to color stain all of the organ systems of the body by injecting colored dyes into the blood stream, but fell short when the dye did not reach the brain. Some time later, his student Edwin Goldman successfully dyed the brain but nothing else when he injected dye into the fluid that surrounds the brain. Their

combined endeavors illuminated the existence of the then undiscovered blood-brain barrier, a protective network of blood vessels and cells that prevents many substances from entering brain tissue and the central nervous system.

Today, the blood-brain barrier remains an innate and vital defender of human health that simultaneously fascinates and frustrates scientists. While it prevents deadly pathogens from reaching the brain, it thwarts the attempts of neuroscientists like Brian K. Kaspar, PhD, and colleagues in the Center for Gene Therapy at The Research Institute who try to develop therapies for childhood disease by targeting the brain and spinal cord more efficiently.

“Nearly all large-molecule drugs and 98 percent of small-molecule drugs do not cross the blood-brain barrier, thus limiting the development of drugs for many central nervous system disorders,” said Dr. Kaspar.

The same difficulty applies to gene delivery strategies. Previous gene therapy efforts to treat two of the most common motor neuron diseases, spinal muscular atrophy (SMA) and ALS (Lou Gehrig’s disease), have either

Bypassing the Brain’s BarrierA NeW sTRATeGy evADes A BIOLOGICAL OBsTACLe TO

GeNe THeRAPy suCCess

Brian Kaspar, PhD

failed to bypass the blood-brain barrier or are clinically impractical since they require interventions considered too numerous or too complicated.

Yet Dr. Kaspar and his team have developed a relatively non-invasive gene delivery method that could lead to impressive new therapies for these disorders, by penetrating the protective blood-brain barrier.

A WINDOW Of OPPORTuNITy Their gene therapy method is detailed in a study featured in Nature Biotechnology in which Dr. Kaspar and colleagues, postdoctoral fellow Dr. Kevin Foust and student Emily Nurre, used AAV9, a subtype of the adeno-associated virus, as the transfer vehicle. AAV vectors have shown promise in several recent clinical trials for neurological disorders, and newly discovered subtypes have shown widespread transfer throughout multiple tissues, such as skeletal and cardiac muscle.

Using a single intravenous injection, researchers delivered AAV9 along with a green fluorescent protein and examined its potential in reaching the central nervous system in neonatal and adult mouse models. Within 10 days, the fluorescent green protein was being expressed along the spinal cord and into the brain with unmatched efficiency.

“The number and extensive distribution of infected cells was utterly remarkable because no one had shown this in the central nervous system from a single injection,” said Dr. Foust.

Although the blood-brain barrier was bypassed in both the neonatal and adult models, the areas in which the fluorescent green protein was expressed differed. In the neonatal model, the vec-tor preferentially targeted basic nerve cells (neurons). In adults it more often targeted astrocytes, the largest and most

The ability to cross the blood brain barrier and globally infect the central nervous system opens up the possibility for countless new avenues of investigation for both basic science and therapeutic endeavors. It’s really quite exciting.– DR. BRIAN K. KAsPAR

common cell type in the central nervous system. Dr. Kaspar says that these differ-ences suggest there is a developmental period during which viral access to these cell populations becomes restricted. “The shift in type of cells targeted is clinically relevant because different dis-orders have different cells participating in the disease process,” said Dr. Kaspar. “For example in the case of SMA, we have to get the virus to neurons in the spinal cord early, and therefore have to treat early. Fortunately, there appears to be a window of time during pediatric de-velopment before the gates of the blood brain barrier become restricted. Addi-tionally, in SMA there are new programs to detect the disease in newborns as part of a newborn screening program, being developed at Nationwide Children’s Hospital and The Ohio State University, so we believe we have a strategy to treat these patients.”

A vIRAL MeTHOD Of evAsION The precise mechanism by which AAV9 penetrates the blood-brain barrier and facilitates gene transfer will require fur-ther study. The predominant astrocyte transduction in adults suggests that AAV9 escapes the brain’s network of blood vessels in a similar manner as it escapes them in skeletal and cardiac muscles, says

Dr. Kaspar. The team hypothesizes that AAV9 is likely enlisting other mecha-nisms to cross the blood-brain barrier such as making use of transport proteins.

exPeCTeD CLINICAL ReLevANCy Despite the need for further study, researchers believe this relatively non-invasive method could have important implications for gene therapy treatments in SMA and ALS. A suggested therapeu-tic for SMA is an increased expression of the survival motor neuron gene; this study’s single injection method could potentially achieve this outcome. Astro-cytes, which were efficiently targeted in both the neonatal and adult models in this study, have been specifically linked to disease progression in ALS.

Additionally, new techniques stemming from this AAV9 research could target neurons in adults. Such strategies could provide new treatment options for diseases such as Huntington’s that involve multiple brain regions. “The ability to cross the blood brain barrier and globally infect the central nervous system opens up the possibility for countless new avenues of investigation for both basic science and therapeutic endeavors,” said Dr. Kaspar. “It’s really quite exciting.”

Injection of a gene therapy virus called AAV9 into the bloodstream of mice results in widespread infection through-out the brain. Purkinje cells, shown in green, are located in the cerebellum and are some of the largest cells in the brain. About 70 percent of Purkinje cells were successfully targeted by this gene therapy strategy.

A risk-assessment technology developed by investigators and programmers at Nationwide Children’s Hospital is helping physicians and

patients stay connected.

Health eTouch is a web-based self-reporting tablet computer that can help clinicians gather critical health information before a patient’s appointment. Patients are given the Health eTouch tablet while in the waiting room to answer multiple choice questions about substance abuse, injury risk, depression and suicidality.

A study in Pediatrics showed that adolescents who participated in computerized screening with real-time results were more likely to be recognized as having high-risk behaviors by their pediatrician than adoles-cents whose screening results were delayed. As a result, physicians can immediately begin appropriate treatment or counseling for their adolescent patients.

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Web exclusive To view a video about how Health eTouch is transforming patient-to-clinician communication, visit us online at www.NationwideChildrens.org − keyword search: Health eTouch.

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