Adeno-associated Viral Vector Delivery Research Articles

Insights into the role of RD3 in guanylate cyclase trafficking, photoreceptor degeneration, and Leber congenital amaurosis.

Retinal degeneration 3 (RD3) is an evolutionarily conserved 23 kDa protein expressed in rod and cone photoreceptor cells. Mutations in the gene encoding RD3 resulting in unstable non-functional C-terminal truncated proteins are responsible for early onset photoreceptor degeneration in Leber Congenital Amaurosis 12 patients, the rd3 mice, and the rcd2 collies. Recent studies have shown that RD3 interacts with guanylate cyclases GC1 and GC2 in retinal cell extracts and HEK293 cells co-expressing GC and RD3. This interaction inhibits GC catalytic activity and promotes the exit of GC1 and GC2 from the endoplasmic reticulum and their trafficking to photoreceptor outer segments. Adeno-associated viral vector delivery of the normal RD3 gene to photoreceptors of the rd3 mouse restores GC1 and GC2 expression and outer segment localization and leads to the long-term recovery of visual function and photoreceptor cell survival. This review focuses on the genetic and biochemical studies that have provided insight into the role of RD3 in photoreceptor function and survival.

Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery

Clinical data support the feasibility and safety of adeno-associated viral (AAV) vectors in gene therapy applications. Despite several clinical trials of AAV-based gene transfer for hemophilia B, a unique set of obstacles impede the development of a similar approach for hemophilia A. These include (i) the size of the factor VIII (fVIII) transgene, (ii) humoral immune responses to fVIII, (iii) inefficient biosynthesis of human fVIII, and (iv) AAV vector immunity. Through bioengineering approaches, a novel fVIII molecule, designated ET3, was developed and shown to improve biosynthetic efficiency 10- to 100-fold. In this study, the utility of ET3 was assessed in the context of liver-directed, AAV-mediated gene transfer into hemophilia A mice. Due to the large size of the expression cassette, AAV-ET3 genomes packaged into viral particles as partial genome fragments. Despite this potential limitation, a single peripheral vein administration of AAV-ET3 into immune-competent hemophilia A mice resulted in correction of the fVIII deficiency at lower vector doses than previously reported for similarly oversized AAV-fVIII vectors. Therefore, ET3 appears to improve vector potency and mitigate at least one of the critical barriers to AAV-based clinical gene therapy for hemophilia A.

In Vivo Adeno-Associated Viral Vector–Mediated Genetic Engineering of White and Brown Adipose Tissue in Adult Mice

Adipose tissue is pivotal in the regulation of energy homeostasis through the balance of energy storage and expenditure and as an endocrine organ. An inadequate mass and/or alterations in the metabolic and endocrine functions of adipose tissue underlie the development of obesity, insulin resistance, and type 2 diabetes. To fully understand the metabolic and molecular mechanism(s) involved in adipose dysfunction, in vivo genetic modification of adipocytes holds great potential. Here, we demonstrate that adeno-associated viral (AAV) vectors, especially serotypes 8 and 9, mediated efficient transduction of white (WAT) and brown adipose tissue (BAT) in adult lean and obese diabetic mice. The use of short versions of the adipocyte protein 2 or uncoupling protein-1 promoters or micro-RNA target sequences enabled highly specific, long-term AAV-mediated transgene expression in white or brown adipocytes. As proof of concept, delivery of AAV vectors encoding for hexokinase or vascular endothelial growth factor to WAT or BAT resulted in increased glucose uptake or increased vessel density in targeted depots. This method of gene transfer also enabled the secretion of stable high levels of the alkaline phosphatase marker protein into the bloodstream by transduced WAT. Therefore, AAV-mediated genetic engineering of adipose tissue represents a useful tool for the study of adipose pathophysiology and, likely, for the future development of new therapeutic strategies for obesity and diabetes.

Dystrophin rescue by trans -splicing: a strategy for DMD genotypes not eligible for exon skipping approaches

RNA-based therapeutic approaches using splice-switching oligonucleotides have been successfully applied to rescue dystrophin in Duchenne muscular dystrophy (DMD) preclinical models and are currently being evaluated in DMD patients. Although the modular structure of dystrophin protein tolerates internal deletions, many mutations that affect nondispensable domains of the protein require further strategies. Among these, trans-splicing technology is particularly attractive, as it allows the replacement of any mutated exon by its normal version as well as introducing missing exons or correcting duplication mutations. We have applied such a strategy in vitro by using cotransfection of pre–trans-splicing molecule (PTM) constructs along with a reporter minigene containing part of the dystrophin gene harboring the stop-codon mutation found in the mdx mouse model of DMD. Optimization of the different functional domains of the PTMs allowed achieving accurate and efficient trans-splicing of up to 30% of the transcript encoded by the cotransfected minigene. Optimized parameters included mRNA stabilization, choice of splice site sequence, inclusion of exon splice enhancers and artificial intronic sequence. Intramuscular delivery of adeno-associated virus vectors expressing PTMs allowed detectable levels of dystrophin in mdx and mdx4Cv, illustrating that a given PTM can be suitable for a variety of mutations.

Hypothalamic proteoglycan syndecan-3 is a novel cocaine addiction resilience factor

Proteoglycans like syndecan-3 have complex signaling roles in addition to their function as structural components of the extracellular matrix. Here, we show that syndecan-3 in the lateral hypothalamus has an unexpected new role in limiting compulsive cocaine intake. In particular, we observe that syndecan-3 null mice self-administer greater amounts of cocaine than wild-type mice. This effect can be rescued by re-expression of syndecan-3 in the lateral hypothalamus with an adeno-associated viral vector. Adeno-associated viral vector delivery of syndecan-3 to the lateral hypothalamus also reduces motivation for cocaine in normal mice. Syndecan-3 limits cocaine intake by modulating the effects of glial-cell-line-derived neurotrophic factor, which uses syndecan-3 as an alternative receptor. Our findings indicate syndecan-3-dependent signaling as a novel therapeutic target for the treatment of cocaine addiction.

Mir‐142‐3p target sequences reduce transgene‐directed immunogenicity following intramuscular adeno‐associated virus 1 vector‐mediated gene delivery

Muscle represents an important tissue target for adeno-associated virus (AAV) vector-mediated gene transfer in muscular, metabolic or blood-related genetic disorders. However, several studies have demonstrated the appearance of immune responses against the transgene product after intramuscular AAV vector delivery that resulted in a limited efficacy of the treatment. Use of microRNAs that are specifically expressed in antigen-presenting cells (APCs) is a promising approach for avoiding those immune responses. Cellular mir-142-3p, which is APC-specific, is able to repress the translation of its target cellular transcripts by binding to a specific target sequences. In the present study, we explored the potential of mir-142-3p specific target sequences with respect to reducing or abolishing immune responses directed against ovalbumin (OVA), a highly immunogenic protein, expressed as transgene and delivered by AAV1 vector administered intramuscularly. The occurrence of immune responses against OVA transgene following intramuscular delivery by AAV have been described previously and resulted in the loss of OVA protein expression. In the present study, we demonstrate that OVA protein expression was maintained when mir-142-3pT sequences were incorporated into the expression cassette. The sustained expression of OVA protein over time correlated with a reduced increase in anti-OVA antibody levels. Furthermore, no cellular infiltrates were observed in the muscle tissue when AAV1 vectors containing four or eight repeats of mir-142-3p target sequences after the OVA sequence were used. The rising humoral and cellular immune responses against OVA protein after intramuscular delivery can be efficiently reduced by the use of mir-142-3p target sequences.

Outer retinal transduction can be achieved after intravitreal delivery of adeno-associated virus serotype 2 vector with glycosidic enzymes

BackgroundGene therapies for retinal disorders, including in current clinical trials, so far have relied on subretinal delivery of adeno-associated virus (AAV) vectors carrying therapeutic DNA into outer retinal cells. Subretinal injection has many limitations over the less-invasive route of administration into the vitreal cavity. However, at present only limited retinal transduction can be achieved after intravitreal delivery of AAV vectors. We hypothesise that the inner limiting membrane and extracellular matrix proteoglycans act as a barrier to AAV vector entry into and movement across the retina. Therefore, glycosidic enzymes, which degrade these extracellular barriers, can improve retinal gene therapy. In this study we investigated the effects of enzymatic digestion of extracellular matrices on the depth of vector penetration into the retina. MethodsThe green fluorescent protein (GFP)-expressing AAV serotype 2 (AAV2) vector was co-injected intravitreally with glycosidic enzymes at their optimum concentration. Efficacy of virus transduction was assessed by visualising fluorescence in histological cross-sections with fluorescence microscopy. We also analysed safety of these treatments and retinal function using electroretinography. FindingsGlycosidic enzymes led to a significant improvement in retinal transduction after intravitreal delivery of AAV2. These enzymes markedly improved transduction of the outer retina, including photoreceptor cells. Electroretinograms were unchanged (compared with controls) even at much higher doses of enzymes than were needed for optimum retinal transduction. InterpretationAAV2-mediated retinal transduction is improved by co-injection of glycosidic enzymes into the vitreous. Improved transduction efficiency may allow intravitreal injection to become the preferred route for delivering gene therapy to the inner and outer retina in both preclinical and clinical settings. FundingUK Medical Research Council.

Visualization of spinal afferent innervation in the mouse colon by AAV8‐mediated GFP expression

Primary afferent neurons whose cell bodies reside in thoracolumbar and lumbosacral dorsal root ganglia (DRG) innervate colon and transmit sensory signals from colon to spinal cord under normal conditions and conditions of visceral hypersensitivity. Histologically, these extrinsic afferents cannot be differentiated from intrinsic fibers of enteric neurons because all known markers label neurons of both populations. Adeno-associated virus (AAV) vectors are capable of transducing DRG neurons after intrathecal administration. We hypothesized that AAV-driven overexpression of green fluorescent protein (GFP) in DRG would enable visualization of extrinsic spinal afferents in colon separately from enteric neurons. Recombinant AAV serotype 8 (rAAV8) vector carrying the GFP gene was delivered via direct lumbar puncture. Green fluorescent protein labeling in DRG and colon was examined using immunohistochemistry. Analysis of colon from rAAV8-GFP-treated mice demonstrated GFP-immunoreactivity (GFP-ir) within mesenteric nerves, smooth muscle layers, myenteric plexus, submucosa, and mucosa, but not in cell bodies of enteric neurons. Notably, GFP-ir colocalized with CGRP and TRPV1 in mucosa, myenteric plexus, and globular-like clusters surrounding nuclei within myenteric ganglia. In addition, GFP-positive fibers were observed in close association with blood vessels of mucosa and submucosa. Analysis of GFP-ir in thoracolumbar and lumbosacral DRG revealed that levels of expression in colon and L6 DRG appeared to be related. These results demonstrate the feasibility of gene transfer to mouse colonic spinal sensory neurons using intrathecal delivery of AAV vectors and the utility of this approach for histological analysis of spinal afferent nerve fibers within colon.

P3‐002: Overexpression of tau in the mouse forebrain using adeno‐associated virus (AAV) leads to tau hyperphosphorylation, formation of neurofibrillary tangles and neurodegeneration

The use of viral vectors provides an alternative approach to replicate cardinal features of tauopathies in the mouse brain and complement existing transgenic mouse models. AAV vectors were designed for overexpression of various forms of human tau, and injected in the lateral ventricle to induce tau pathology in the mouse forebrain. We generated AAV constructs encoding various forms of the human tau protein under the control of the mouse PGK-1 promoter. Two wild-type versions of human tau (tau4R0N, tau3R0N) were compared with a tau mutant causing frontotemporal dementia (tau4R0N-P301S), and an aggregation-deficient mutated variant (tau4R0N-I277P/I308P). AAV serotype 6 vectors were bilaterally injected in the lateral ventricles of mouse neonates using a stereotactic system (ICV injections). In order to compare the kinetic of the pathology induced by these different forms of tau, the injected mice were analyzed at 6, 9 and 24 weeks post-injection. We monitored by histological analysis the deposition of phosphorylated and aggregated forms of tau in the mouse cortex and hippocampus, as well as the induced neurodegeneration. Changes in animal motor performance were assessed behaviorally (rotarod test). AAV-injected mice displayed a clear neuronal accumulation of human tau protein in various brain regions including the cerebral cortex, hippocampus and brainstem. Expectedly, high levels of human tau were predominantly found in brain regions lining the ventricular system. At an early time point, we showed significant accumulation of hyperphosphorylated tau protein (AT8 staining) in the neuronal cell bodies as well as in the dendritic compartment. Tau aggregation was revealed by immunohistochemistry using a conformational antibody and confirmed by Gallyas staining at later time points. A significant neurodegeneration was also observed, mostly in the region of the cerebral cortex close to the site of vector injection. In addition, the motor performance of the AAV-injected mice became progressively impaired. ICV delivery of AAV vectors in mouse neonates can efficiently induce neuronal tau expression in the forebrain. The resulting accumulation of human tau leads to a robust neuropathology mainly characterized by tau aggregation. The observed pathology correlates with acute neurodegeneration and progressive impairment of animal motor behavior.

AAV-based neonatal gene therapy for hemophilia A: long-term correction and avoidance of immune responses in mice

Hemophilia A gene therapy has been hampered by immune responses to vector-associated antigens and by neutralizing antibodies or inhibitors to the factor VIII (FVIII) protein; these ‘inhibitors’ more commonly effect hemophilia A patients than those with hemophilia B. A gene replacement strategy beginning in the neonatal period may avoid the development of these immune responses and lead to prolonged expression with correction of phenotype thereby avoiding long-term consequences. Serotype rh10 AAV was developed splitting the FVIII coding sequence into heavy and light chains with the chicken β-actin promoter/CMV enhancer for dual recombinant AAV vector delivery. Coinjection of virions of each FVIII chain intravenously to mice on the second day of life was performed. Mice express sustained FVIII antigen levels of ≥5% to 22 months of life without the development of antibodies to FVIII. Phenotypic correction was manifest in all AAV-FVIII-treated mice as demonstrated by functional assay and reduction in bleeding time. This study demonstrates the use of AAV in a gene replacement strategy in neonatal mice that establishes both long-term phenotypic correction of hemophilia A and lack of antibody development to FVIII in this disease model where AAV is administered shortly after birth. These studies support consideration of gene replacement therapy for diseases that are diagnosed in utero or in the early neonatal period.

Magnetically enhanced adeno-associated viral vector delivery for human neural stem cell infection

Gene therapy technology is a powerful tool to elucidate the molecular cues that precisely regulate stem cell fates, but developing safe vehicles or mechanisms that are capable of delivering genes to stem cells with high efficiency remains a challenge. In this study, we developed a magnetically guided adeno-associated virus (AAV) delivery system for gene delivery to human neural stem cells (hNSCs). Magnetically guided AAV delivery resulted in rapid accumulation of vectors on target cells followed by forced penetration of the vectors across the plasma membrane, ultimately leading to fast and efficient cellular transduction. To combine AAV vectors with the magnetically guided delivery, AAV was genetically modified to display hexa-histidine (6xHis) on the physically exposed loop of the AAV2 capsid (6xHis AAV), which interacted with nickel ions chelated on NTA-biotin conjugated to streptavidin-coated superparamagnetic iron oxide nanoparticles (NiStNPs). NiStNP-mediated 6xHis AAV delivery under magnetic fields led to significantly enhanced cellular transduction in a non-permissive cell type (i.e., hNSCs). In addition, this delivery method reduced the viral exposure times required to induce a high level of transduction by as much as to 2–10 min of hNSC infection, thus demonstrating the great potential of magnetically guided AAV delivery for numerous gene therapy and stem cell applications.

The AAV9 receptor and its modification to improve in vivo lung gene transfer in mice

Vectors based on adeno-associated virus (AAV) serotype 9 are candidates for in vivo gene delivery to many organs, but the receptor(s) mediating these tropisms have yet to be defined. We evaluated AAV9 uptake by glycans with terminal sialic acids (SAs), a common mode of cellular entry for viruses. We found, however, that AAV9 binding increased when terminal SA was enzymatically removed, suggesting that galactose, which is the most commonly observed penultimate monosaccharide to SA, may mediate AAV9 transduction. This was confirmed in mutant CHO Pro-5 cells deficient in the enzymes involved in glycoprotein biogenesis, as well as lectin interference studies. Binding of AAV9 to glycans with terminal galactose was demonstrated via glycan binding assays. Co-instillation of AAV9 vector with neuraminidase into mouse lung resulted in exposure of terminal galactose on the apical surface of conducting airway epithelial cells, as shown by lectin binding and increased transduction of these cells, demonstrating the possible utility of this vector in lung-directed gene transfer. Increasing the abundance of the receptor on target cells and improving vector efficacy may improve delivery of AAV vectors to their therapeutic targets.

Retroviral Vectors Induce Epigenetic Chromatin Modifications and IL-10 Production in Transduced B Cells via Activation of Toll-like Receptor 2

The immune response toward viral vectors used for gene therapy and genetic vaccination appears to be critically important in determining the therapeutic outcome. However, the mechanisms that control the immune response following gene transfer are poorly understood. Unexpectedly, we found that integrating retroviral vector particles induce stable interleukin-10 (IL-10) production in murine (BALB/c H-2(d)) transduced B cells. This requires a novel mechanism whereby the interaction of retroviral vector particle with its cognate cellular receptor activates intracellular signaling pathways resulting in stable epigenetic modifications. Murine B cells exposed to retroviral vector particles triggered the colocalization of the retroviral cellular receptor [mouse cationic amino acid transporter 1 (mCAT1)] and Toll-like receptor 2 (TLR2) into lipid microrafts, which in turn activated TLR2 signaling pathways. TLR2 activation induced STAT3 phosphorylation and increased phosphorylated histone 3 (H3) at the STAT3-binding site of the IL-10 promoter. In addition, TLR2 activation during transduction activates nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (NFKBIA), thereby preventing the translocation of the nuclear factor-κB (NF-κB) complex to the nucleus and the transcription of proinflammatory cytokines. These findings open new perspectives for controlling immune responses following gene therapy and genetic vaccination.

Gene therapy for glaucoma

Glaucoma is a chronic progressive disease for which ideal treatment would provide a localized long-lasting therapy with minimal side-effects. A gene therapy approach in which a mutated gene is replaced or inactivated, or in which a new gene is introduced, could provide a novel and more effective way of targeting the disease. The aim of this review is to provide an update on recent advances in the field of gene therapy for the treatment of glaucoma. Both viral and nonviral vector gene delivery systems have been used to target specific tissues involved in the pathogenesis of glaucoma. These tissues include the trabecular meshwork, ciliary body, ciliary epithelium, Müller cells, and retinal ganglion cells. Recent studies in large animal models have demonstrated effective long-term gene expression in the trabecular meshwork following intracameral delivery of adeno-associated viral vectors and lentiviral vectors with limited effect on surrounding ocular tissues. Other promising studies have focused on vector-mediated expression of neurotrophic factors and have demonstrated a neuroprotective effect following intravitreal delivery of vectors in glaucomatous animal models. Given the recent laboratory studies utilizing gene therapy techniques to lower intraocular pressure and to provide neuroprotection, and the continued development of tissue-specific vectors, it seems that we are well poised for a new generation of treatments for glaucoma.

Fasting Increases the In Vivo Gene Delivery of AAV Vectors

Successful gene therapy of many genetic diseases requires efficient delivery of the gene to several tissues of the organism. Adeno-associated virus (AAV) is, to date, the sole vehicle that allows to achieving this result but only at the condition of administering very large amounts of vectors. This, however, raises questions about the feasibility of the large-scale production and about the safety of the approach. One way to overcome both problems would be to develop strategies that increase the in vivo efficiency. Here, we investigated the effect of fasting on the transduction efficiency of AAV serotypes 2, 6, and 9. The transgene expression was followed for several weeks and vector biodistribution was determined by real-time polymerase chain reaction (PCR) . The results show that fasting increases the transduction efficiency of all three serotypes. Altogether, we present here a simple and clinically acceptable approach that may allow to reducing the vector dose.

Proteasome Inhibitors Enhance Gene Delivery by AAV Virus Vectors Expressing Large Genomes in Hemophilia Mouse and Dog Models: A Strategy for Broad Clinical Application

Delivery of genes that are larger than the wild-type adeno-associated virus (AAV) 4,681 nucleotide genome is inefficient using AAV vectors. We previously demonstrated in vitro that concurrent proteasome inhibitor (PI) treatment improves transduction by AAV vectors encoding oversized transgenes. In this study, an AAV vector with a 5.6 kilobase (kb) factor VIII expression cassette was used to test the effect of an US Food and Drug Administration-approved PI (bortezomib) treatment concurrent with vector delivery in vivo. Intrahepatic vector delivery resulted in factor VIII expression that persisted for >1 year in hemophilia mice. Single-dose bortezomib given with AAV2 or AAV8 factor VIII vector enhanced expression on average ~600 and ~300%, respectively. Moreover, coadministration of AAV8.canineFVIII (1 × 10(13) vg/kg) and bortezomib in hemophilia A dogs (n = 4) resulted in normalization of the whole blood clotting time (WBCT) and 90% reduction in hemorrhages for >32 months compared to untreated hemophilia A dogs (n = 3) or dogs administered vector alone (n = 3). Demonstration of long-term phenotypic correction of hemophilia A dogs with combination adjuvant bortezomib and AAV vector expressing the oversized transgene establishes preclinical studies that support testing in humans and provides a working paradigm to facilitate a significant expansion of therapeutic targets for human gene therapy.

Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B

AbstractMuscle represents an important tissue target for adeno-associated viral (AAV) vector-mediated gene transfer of the factor IX (FIX) gene in hemophilia B (HB) subjects with advanced liver disease. Previous studies of direct intramuscular administration of an AAV-FIX vector in humans showed limited efficacy. Here we adapted an intravascular delivery system of AAV vectors encoding the FIX transgene to skeletal muscle of HB dogs. The procedure, performed under transient immunosuppression (IS), resulted in widespread transduction of muscle and sustained, dose-dependent therapeutic levels of canine FIX transgene up to 10-fold higher than those obtained by intramuscular delivery. Correction of bleeding time correlated clinically with a dramatic reduction of spontaneous bleeding episodes. None of the dogs (n = 14) receiving the AAV vector under transient IS developed inhibitory antibodies to canine FIX; transient inhibitor was detected after vector delivery without IS. The use of AAV serotypes with high tropism for muscle and low susceptibility to anti-AAV2 antibodies allowed for efficient vector administration in naive dogs and in the presence of low- but not high-titer anti-AAV2 antibodies. Collectively, these results demonstrate the feasibility of this approach for treatment of HB and highlight the importance of IS to prevent immune responses to the FIX transgene product.

AAV Provides an Alternative for Gene Therapy of the Peripheral Sensory Nervous System

Recombinant adeno-associated virus (rAAV) is a promising vector for applications in the central nervous system (CNS). Clinical phase I/II studies have demonstrated safety of rAAV-expressing therapeutic transgenes for a variety of benign (i.e., noncancer) CNS disorders, namely, Canavan disease,1 Parkinson’s disease,2,3,4 and Batten disease.5 Human studies were also recently reported targeting the retina.6,8,9,9 From the early stages of preclinical development, rAAV was appealing because it was found to be neurotrophic and capable of long-term activity without immune interference or toxicity when injected into the CNS.10

The Pleiotropic Effects of Natural AAV Infections on Liver-directed Gene Transfer in Macaques

Adeno-associated viral (AAV) vectors hold great potential for liver-directed gene therapy. Stable and high levels of transgene expression have been achieved in many murine models. Systemic delivery of AAV vectors in nonhuman primates (NHPs) that are natural hosts of AAVs appear to be challenging due to the high prevalence of pre-existing neutralizing antibodies (NAbs). This study evaluates the performance of AAV8, hu.37, and rh.8 vectors expressing green fluorescent protein (GFP) from a liver-specific promoter in rhesus macaques. Two of the animals that received AAV8 showed transduction of 24 and 40% of hepatocytes 7 days after systemic vector delivery. Importantly, expression was detected in several animals after 35 days despite the elevation of liver enzymes and development of transgene-specific T cells in liver. Pre-existing low levels of NAbs profoundly impacted the outcome of gene transfer and redirected vector DNA to spleen. We developed a sensitive in vivo passive transfer assay to detect low levels of NAbs to these novel AAV serotypes. Other strategies need to be developed to reduce immune response to the transgene in order to maintain long-term gene expression.

Efficient gene transfer to muscle satellite cells by in utero intravenous delivery of adeno-associated viral vector

Stitelman, David H. MD; Bora, Archana MS; Jeremy, Traas PhD; Merianos, Demetri MD; Flake, Alan W. MD, FACS; Brazelton, Tim MD, PhD Author Information