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1.  Increased Mucosal CD4+ T Cell Activation in Rhesus Macaques following Vaccination with an Adenoviral Vector 
Journal of Virology  2014;88(15):8468-8478.
The possibility that vaccination with adenovirus (AdV) vectors increased mucosal T cell activation remains a central hypothesis to explain the potential enhancement of HIV acquisition within the Step trial. Modeling this within rhesus macaques is complicated because human adenoviruses, including human adenovirus type 5 (HAdV-5), are not endogenous to macaques. Here, we tested whether vaccination with a rhesus macaque-derived adenoviral vector (simian adenovirus 7 [SAdV-7]) enhances mucosal T cell activation within rhesus macaques. Following intramuscular SAdV-7 vaccination, we observed a pronounced increase in SAdV-7-specific CD4+ T cell responses in peripheral blood and, more dramatically, in rectal mucosa tissue. Vaccination also induced a significant increase in the frequency of activated memory CD4+ T cells in SAdV-7- and HAdV-5-vaccinated animals in the rectal mucosa but not in peripheral blood. These fluctuations within the rectal mucosa were also associated with a pronounced decrease in the relative frequency of naive resting CD4+ T cells. Together, these results indicate that peripheral vaccination with an AdV vector can increase the activation of mucosal CD4+ T cells, potentially providing an experimental model to further evaluate the role of host-vector interactions in increased HIV acquisition after AdV vector vaccination.
IMPORTANCE The possibility that vaccination with a human adenovirus 5 vector increased mucosal T cell activation remains a central hypothesis to explain the potential enhancement of human immunodeficiency virus (HIV) acquisition within the Step trial. In this study, we tested whether vaccination with a rhesus macaque-derived adenoviral vector in rhesus macaques enhances mucosal CD4+ T cell activation, the main cell target of simian immunodeficiency virus (SIV)/HIV. The results showed that vaccination with an adenoviral vector indeed increases activation of mucosal CD4+ T cells and potentially increases susceptibility to SIV infection.
PMCID: PMC4135938  PMID: 24829340
2.  The First Journal on Human Gene Therapy Celebrates its 25th Anniversary 
Human Gene Therapy  2014;25(1):1-2.
PMCID: PMC3900113  PMID: 24444178
3.  Biodistribution of AAV8 Vectors Expressing Human Low-Density Lipoprotein Receptor in a Mouse Model of Homozygous Familial Hypercholesterolemia 
Recombinant adeno-associated viral vectors based on serotype 8 (AAV8) transduce liver with superior tropism following intravenous (IV) administration. Previous studies conducted by our lab demonstrated that AAV8-mediated transfer of the human low-density lipoprotein receptor (LDLR) gene driven by a strong liver-specific promoter (thyroxin-binding globulin [TBG]) leads to high level and persistent gene expression in the liver. The approach proved efficacious in reducing plasma cholesterol levels and resulted in the regression of atherosclerotic lesions in a murine model of homozygous familial hypercholesterolemia (hoFH). Prior to advancing this vector, called AAV8.TBG.hLDLR, to the clinic, we set out to investigate vector biodistribution in an hoFH mouse model following IV vector administration to assess the safety profile of this investigational agent. Although AAV genomes were present in all organs at all time points tested (up to 180 days), vector genomes were sequestered mainly in the liver, which contained levels of vector 3 logs higher than that found in other organs. In both sexes, the level of AAV genomes gradually declined and appeared to stabilize 90 days post vector administration in most organs although vector genomes remained high in liver. Vector loads in the circulating blood were high and close to those in liver at the early time point (day 3) but rapidly decreased to a level close to the limit of quantification of the assay. The results of this vector biodistribution study further support a proposed clinical trial to evaluate AAV8 gene therapy for hoFH patients.
PMCID: PMC4003465  PMID: 24070336
4.  Vector Sequences Are Not Detected in Tumor Tissue from Research Subjects with Ornithine Transcarbamylase Deficiency Who Previously Received Adenovirus Gene Transfer 
Human Gene Therapy  2013;24(9):814-819.
A 66-year-old woman heterozygous for a mutation in the ornithine transcarbamylase gene (Otc) participated in a phase I gene therapy trial for OTC deficiency. She received an adenovirus (Ad) vector expressing the functional OTC gene by intraportal perfusion. Fourteen years later she developed and subsequently died of hepatocellular carcinoma. A second subject, a 45-year-old woman, enrolled in the same trial presented with colon cancer 15 years later. We sought to investigate a possible association between the development of a tumor and prior adenoviral gene transfer in these two subjects. We developed and validated a sensitive nested polymerase chain reaction assay for recovering recombinant Ad sequences from host tissues. Using this method, we could not detect any Ad vector DNA in either tumor or normal tissue from the two patients. Our results are informative in ruling out the possibility that the adenoviral vector might have contributed to the development of cancer in those two subjects.
Zhong and colleagues use a nested PCR assay to examine whether adenoviral gene transfer led to the development of cancer in two clinical research subjects with ornithine transcarbamylase deficiency. Using this approach, they were unable to detect adenovirus vector genomes in normal or tumor tissues from the subjects more than a decade after vector infusion. These results suggest that adenoviral gene transfer was an unlikely contributor to the colon cancer or hepatocellular carcinoma observed in the two research subjects.
PMCID: PMC3768231  PMID: 24010702
5.  Enhancing the Utility of Adeno-Associated Virus Gene Transfer through Inducible Tissue-Specific Expression 
Human Gene Therapy Methods  2013;24(4):270-278.
The ability to regulate both the timing and specificity of gene expression mediated by viral vectors will be important in maximizing its utility. We describe the development of an adeno-associated virus (AAV)-based vector with tissue-specific gene regulation, using the ARGENT dimerizer-inducible system. This two-vector system based on AAV serotype 9 consists of one vector encoding a combination of reporter genes from which expression is directed by a ubiquitous, inducible promoter and a second vector encoding transcription factor domains under the control of either a heart- or liver-specific promoter, which are activated with a small molecule. Administration of the vectors via either systemic or intrapericardial injection demonstrated that the vector system is capable of mediating gene expression that is tissue specific, regulatable, and reproducible over induction cycles. Somatic gene transfer in vivo is being considered in therapeutic applications, although its most substantial value will be in basic applications such as target validation and development of animal models.
Chen and colleagues describe the development of an AAV-based vector with tissue-specific gene regulation, using the ARGENT dimerizer-inducible system. They demonstrate that administration of these vectors via either systemic or intrapericardial injection leads to gene expression that is tissue specific, regulatable, and reproducible over induction cycles.
PMCID: PMC3753727  PMID: 23895325
7.  Lessons Learned from the Clinical Development and Market Authorization of Glybera 
Bryant and colleagues follow the development of Glybera (alipogene tiparvovec), the first gene therapy product approved in the European Union, from early preclinical studies through the approval process. They review key data from human and animal studies with an emphasis on issues that will be critical to other gene therapy products. The article concludes with an analysis of the complex review process that eventually led to Glybera's approval.
PMCID: PMC3992977  PMID: 23808604
8.  Mapping the Structural Determinants Responsible for Enhanced T Cell Activation to the Immunogenic Adeno-Associated Virus Capsid from Isolate Rhesus 32.33 
Journal of Virology  2013;87(17):9473-9485.
Avoiding activation of immunity to vector-encoded proteins is critical to the safe and effective use of adeno-associated viral (AAV) vectors for gene therapy. While commonly used serotypes, such as AAV serotypes 1, 2, 7, 8, and 9, are often associated with minimal and/or dysfunctional CD8+ T cell responses in mice, the threshold for immune activation appears to be lower in higher-order species. We have modeled this discrepancy within the mouse by identifying two capsid variants with differential immune activation profiles: AAV serotype 8 (AAV8) and a hybrid between natural rhesus isolates AAVrh32 and AAVrh33 (AAVrh32.33). Here, we aimed to characterize the structural determinants of the AAVrh32.33 capsid that augment cellular immunity to vector-encoded proteins or those of AAV8 that may induce tolerance. We hypothesized that the structural domain responsible for differential immune activation could be mapped to surface-exposed regions of the capsid, such as hypervariable regions (HVRs) I to IX of VP3. To test this, a series of hybrid AAV capsids was constructed by swapping domains between AAV8 and AAVrh32.33. By comparing their ability to generate transgene-specific T cells in vivo versus the stability of transgene expression in the muscle, we confirmed that the functional domain lies within the VP3 portion of the capsid. Our studies were able to exclude the regions of VP3 which are not sufficient for augmenting the cellular immune response, notably, HVRs I, II, and V. We have also identified HVR IV as a region of interest in conferring the efficiency and stability of muscle transduction to AAVrh32.33.
PMCID: PMC3754105  PMID: 23720715
9.  An AAV Vector-Mediated Gene Delivery Approach Facilitates Reconstitution of Functional Human CD8+ T Cells in Mice 
PLoS ONE  2014;9(2):e88205.
In the present study, a novel adeno-associated virus (AAV) vector-mediated gene delivery approach was taken to improve the reconstitution of functional CD8+ T cells in humanized mice, thereby mimicking the human immune system (HIS). Human genes encoding HLA-A2 and selected human cytokines (A2/hucytokines) were introduced to an immune-deficient mouse model [NOD/SCID/IL2rγnull (NSG) mice] using AAV serotype 9 (AAV9) vectors, followed by transplantation of human hematopoietic stem cells. NSG mice transduced with AAV9 encoding A2/hucytokines resulted in higher levels of reconstitution of human CD45+ cells compared to NSG mice transduced with AAV9 encoding HLA-A2 alone or HLA-A2-transgenic NSG mice. Furthermore, this group of HIS mice also mounted the highest level of antigen-specific A2-restricted human CD8+ T-cell response upon vaccination with recombinant adenoviruses expressing human malaria and HIV antigens. Finally, the human CD8+ T-cell response induced in human malaria vaccine-immunized HIS mice was shown to be functional by displaying cytotoxic activity against hepatocytes that express the human malaria antigen in the context of A2 molecules. Taken together, our data show that AAV vector-mediated gene delivery is a simple and efficient method to transfer multiple human genes to immune-deficient mice, thus facilitating successful reconstitution of HIS in mice. The HIS mice generated in this study should ultimately allow us to swiftly evaluate the T-cell immunogenicity of various human vaccine candidates in a pre-clinical setting.
PMCID: PMC3916402  PMID: 24516613
10.  Adeno-Associated Virus Serotype 8 Gene Therapy Leads to Significant Lowering of Plasma Cholesterol Levels in Humanized Mouse Models of Homozygous and Heterozygous Familial Hypercholesterolemia 
Human Gene Therapy  2012;24(1):19-26.
Familial hypercholesterolemia (FH) is a life-threatening genetic disease caused by mutations in the gene encoding low-density lipoprotein receptor (LDLR). As a bridge to clinical trials, we generated a “humanized” mouse model lacking LDLR and apolipoprotein B (ApoB) mRNA editing catalytic polypeptide-1 (APOBEC-1) expression and expressing a human ApoB100 transgene in order to permit more authentic simulation of in vivo interactions between the clinical transgene product, human LDLR (hLDLR), and its endogenous ligand, human ApoB100. On a chow diet, the humanized LDLR-deficient mice have substantial hypercholesterolemia and a lipoprotein phenotype more closely resembling human homozygous FH (hoFH) than in previous mouse models of FH. On injection of an adeno-associated virus serotype 8 (AAV8) vector encoding the human LDLR cDNA, significant correction of hypercholesterolemia was realized at doses as low as 1.5×1011 genome copies (GC)/kg. Given that some patients with heterozygous FH (heFH) cannot be adequately treated with current therapy, we then extended our studies to similarly “humanized” mice that were heterozygous for LDLR deficiency, and that have a lipoprotein phenotype resembling heterozygous FH. Injection of AAV8-hLDLR brought about significant reduction in total and LDL cholesterol at doses as low as 5×1011 GC/kg. Collectively, these data demonstrate the safety and efficacy of the liver-specific AAV8-hLDLR vector in the treatment of humanized mice modeling both hoFH and heFH.
Kassim and colleagues demonstrate that injection of an adeno-associated virus serotype 8 (AAV8) vector encoding the human low-density lipoprotein receptor (LDLR) cDNA results in significant correction of hypercholesterolemia in humanized mouse models of homozygous and heterozygous familial hypercholesterolemia (FH).
PMCID: PMC3555111  PMID: 22985273
11.  Human Treg responses allow sustained recombinant adeno-associated virus–mediated transgene expression 
The Journal of Clinical Investigation  2013;123(12):5310-5318.
Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases; however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss of efficacy. To determine whether rAAV vector expression can persist long term, we administered rAAV vectors expressing normal, M-type α-1 antitrypsin (M-AAT) to AAT-deficient subjects at various doses by multiple i.m. injections. M-specific AAT expression was observed in all subjects in a dose-dependent manner and was sustained for more than 1 year in the absence of immune suppression. Muscle biopsies at 1 year had sustained AAT expression and a reduction of inflammatory cells compared with 3 month biopsies. Deep sequencing of the TCR Vβ region from muscle biopsies demonstrated a limited number of T cell clones that emerged at 3 months after vector administration and persisted for 1 year. In situ immunophenotyping revealed a substantial Treg population in muscle biopsy samples containing AAT-expressing myofibers. Approximately 10% of all T cells in muscle were natural Tregs, which were activated in response to AAV capsid. These results suggest that i.m. delivery of rAAV type 1–AAT (rAAV1-AAT) induces a T regulatory response that allows ongoing transgene expression and indicates that immunomodulatory treatments may not be necessary for rAAV-mediated gene therapy.
PMCID: PMC3859421  PMID: 24231351
12.  Intramuscular Injection of AAV8 in Mice and Macaques Is Associated with Substantial Hepatic Targeting and Transgene Expression 
PLoS ONE  2014;9(11):e112268.
Intramuscular (IM) administration of adeno-associated viral (AAV) vectors has entered the early stages of clinical development with some success, including the first approved gene therapy product in the West called Glybera. In preparation for broader clinical development of IM AAV vector gene therapy, we conducted detailed pre-clinical studies in mice and macaques evaluating aspects of delivery that could affect performance. We found that following IM administration of AAV8 vectors in mice, a portion of the vector reached the liver and hepatic gene expression contributed significantly to total expression of secreted transgenes. The contribution from liver could be controlled by altering injection volume and by the use of traditional (promoter) and non-traditional (tissue-specific microRNA target sites) expression control elements. Hepatic distribution of vector following IM injection was also noted in rhesus macaques. These pre-clinical data on AAV delivery should inform safe and efficient development of future AAV products.
PMCID: PMC4230988  PMID: 25393537
13.  In Memory of Sonia Skarlatos, PhD (1953–2013) 
Human Gene Therapy  2013;24(11):895.
PMCID: PMC3814981  PMID: 24164235
14.  Analyzing the cancer methylome through targeted bisulfite sequencing 
Cancer letters  2012;340(2):10.1016/j.canlet.2012.10.040.
Bisulfite conversion of genomic DNA combined with next-generation sequencing (NGS) has become a very effective approach for mapping the whole-genome and sub-genome wide DNA methylation landscapes. However, whole methylome shotgun bisulfite sequencing is still expensive and not suitable for analyzing large numbers of human cancer specimens. Recent advances in the development of targeted bisulfite sequencing approaches offer several attractive alternatives. The characteristics and applications of these methods are discussed in this review article. In addition, the bioinformatic tools that can be used for sequence capture probe design as well as downstream sequence analyses are also addressed.
PMCID: PMC3616138  PMID: 23200671
15.  Single Nucleotide Polymorphisms in Cholesteryl Ester Transfer Protein Gene And Recurrent Coronary Heart Disease or Mortality in Patients with Established Atherosclerosis 
The American journal of cardiology  2013;112(9):1287-1292.
It is not known whether genetic variants in the cholesteryl-ester-transfer-protein (CETP) gene are associated with recurrent coronary heart disease events or mortality in secondary prevention patients. Among 3717 acute coronary syndrome (ACS) or coronary artery bypass grafting (CABG) patients enrolled in a prospective genetic registry; we evaluated whether CETP gene variants previously shown to be associated with reduced CETP activity and high-density-lipoprotein-cholesterol increase (“A” allele for both TaqIB [rs708272] and rs12149545) are associated with a reduction in recurrent myocardial infarction [MI], recurrent revascularization or death. At 4.5 years of follow-up; 439 recurrent MI, 698 recurrent revascularizations and 756 deaths occurred. Using an additive model of inheritance, the “A” allele for rs708272 was not associated with recurrent MI (HR 0.95, 95% CI 0.78-1.17 for AG; HR 0.89, 95% CI 0.67-1.19 for AA; compared with GG genotype), recurrent revascularization (HR 1.13, 95% CI 0.95-1.33 for AG; HR 1.05, 95% CI 0.84-1.32 for AA) or mortality (HR 1.02, 95% CI 0.86-1.19 for AG; HR 1.11, 95% CI 0.91-1.37 for AA) in the overall cohort. Similar results were seen for the “A” allele for rs12149545. In the CABG subgroup, AG genotype for rs708272 was associated with an increased mortality (HR 1.38, 95% CI 1.06-1.79) compared to GG genotype. Results remained consistent using dominant model of inheritance. In conclusion, genetic CETP variants were not associated with recurrent MI or recurrent revascularization in overall cohort with a possible mortality increase in CABG patients.
PMCID: PMC3800478  PMID: 23891427
Cholesteryl-ester-transfer-protein; TaqIB; recurrent events; mortality
16.  Muscle-directed Gene Therapy for Hemophilia B with More Efficient and Less Immunogenic AAV Vectors 
Adeno-associated viral vector (AAV)-mediated and muscle-directed gene therapy is a safe and noninvasive approach to treat hemophilia B and other genetic diseases. However, low efficiency of transduction, inhibitor formation and high prevalence of pre-existing immunity to the AAV capsid in humans remain as main challenges for AAV2-based vectors using this strategy. Vectors packaged with AAV7, 8, and 9 serotypes have improved gene transfer efficiencies and may provide potential alternatives to overcome these problems.
To compare the long-term expression of canine factor IX (cFIX) levels and anti-cFIX antibody responses following intramuscular injection of vectors packaged with AAV1, 2, 5, 7, 8, and 9 capsid in immunocompetent hemophilia B mice.
Methods and results
Highest expression was detected in mice injected with AAV2/8 vector (28% of normal), followed by AAV2/9 (15%) and AAV2/7 (10%). cFIX expression by AAV2/1 only ranged from 0–5% of normal levels. High incidences of anti-cFIX inhibitor (IgG) were detected in mice injected with AAV2 and 2/5 vectors, followed by AAV2/1. None of the mice treated with AAV2/7, 2/8, and 2/9 developed inhibitors or capsid T cells.
AAV7, 8, and 9 are more efficient and safer vectors for muscle-directed gene therapy with high levels of transgene expression and absence of inhibitor formation. The absence of antibody response to transgene by AAV7, 8, and 9 is independent of vector dose but may be due to the fact that these three serotypes are associated with high level distribution to, and transduction of, hepatocytes following i.m. injection.
PMCID: PMC3393098  PMID: 21883883
Hemophilia B; gene therapy; adeno-associated viruses (AAV); factor IX; muscle
17.  CpG-depleted adeno-associated virus vectors evade immune detection 
The Journal of Clinical Investigation  2013;123(7):2994-3001.
Due to their efficient transduction potential, adeno-associated virus (AAV) vectors are leading candidates for gene therapy in skeletal muscle diseases. However, immune responses toward the vector or transgene product have been observed in preclinical and clinical studies. TLR9 has been implicated in promoting AAV-directed immune responses, but vectors have not been developed to circumvent this barrier. To assess the requirement of TLR9 in promoting immunity toward AAV-associated antigens following skeletal muscle gene transfer in mice, we compared immunological responses in WT and Tlr9-deficient mice that received an AAV vector with an immunogenic capsid, AAVrh32.33. In Tlr9-deficient mice, IFN-γ T cell responses toward capsid and transgene antigen were suppressed, resulting in minimal cellular infiltrate and stable transgene expression in target muscles. These findings suggest that AAV-directed immune responses may be circumvented by depleting the ligand for TLR9 (CpG sequences) from the vector genome. Indeed, we found that CpG-depleted AAVrh32.33 vectors could establish persistent transgene expression, evade immunity, and minimize infiltration of effector cells. Thus, CpG-depleted AAV vectors could improve outcome of clinical trials of gene therapy for skeletal muscle disease.
PMCID: PMC3696560  PMID: 23778142
18.  Hepatic Gene Transfer in Neonatal Mice by Adeno-Associated Virus Serotype 8 Vector 
Human Gene Therapy  2011;23(5):533-539.
For genetic diseases that manifest at a young age with irreversible consequences, early treatment is critical and essential. Neonatal gene therapy has the advantages of achieving therapeutic effects before disease manifestation, a low vector requirement and high vector-to-cell ratio, and a relatively immature immune system. Therapeutic effects or long-term rescue of neonatal lethality have been demonstrated in several animal models. However, vigorous cell proliferation in the newborn stage is a significant challenge for nonintegrating vectors, such as adeno-associated viral (AAV) vector. Slightly delaying the injection age, and readministration at a later time, are two of the alternative strategies to solve this problem. In this study, we demonstrated robust and efficient hepatic gene transfer by self-complementary AAV8 vector in neonatal mice. However, transduction quickly decreased over a few weeks because of vector dilution caused by fast proliferation. Delaying the injection age improved sustained expression, although it also increased neutralizing antibody (NAb) responses to AAV capsid. This approach can be used to treat genetic diseases with slow progression. For genetic diseases with early onset and severe consequences, early treatment is essential. A second injection of vector of a different serotype at a later time may overcome preexisting NAb and achieve sustained therapeutic effects.
Wang and colleagues conduct a series of preclinical animal studies examining the kinetics of AAV gene transfer. They demonstrate that self-complementary AAV8 results in robust and efficient hepatic gene transfer in neonatal mice. Yet, this transduction quickly decreases over a few weeks because of vector dilution caused by rapid cell proliferation in the liver of growing young mice.
PMCID: PMC3360497  PMID: 22098408
19.  Self-Reactive CFTR T Cells in Humans: Implications for Gene Therapy 
Cystic fibrosis (CF) is one of the most common autosomal recessive lethal disorders affecting white populations of northern European ancestry. To date there is no cure for CF. Life-long treatments for CF are being developed and include gene therapy and the use of small-molecule drugs designed to target specific cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations. Irrespective of the type of molecular therapy for CF, which may include gene replacement, exon skipping, nonsense suppression, or molecular correctors, because all of these modulate gene expression there is an inherent risk of activation of T cells against the wild-type version of CFTR. Here we report the validation of the human interferon-γ enzyme-linked immunospot assay and its application for the analysis of CFTR-specific T cell responses in patients with CF and in non-CF subjects. We found non-CF subjects with low levels of self-reactive CFTR-specific T cells in the United States and several patients with CF with low to high levels of self-reactive CFTR-specific T cells in both the United States and the United Kingdom.
Calcedo and colleagues develop and validate a sensitive interferon-γ enzyme-linked immunospot assay for detection of human T cells specific to the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein. They detect CFTR reactive T cells in a significant portion of CF patients. Remarkably, T cells recognizing CFTR are also identified in non-CF subjects. These data have important implications for measuring and interpreting transgene-specific immune responses in gene therapy trials.
PMCID: PMC4003469  PMID: 23790242
20.  The Black Canyon Forecast Station: Experiences And Lessons Learned 
To evaluate the sociological effect on indigenous biological event signature recognition and community resilience due to the operational activities of an infectious disease forecast station.
The nation’s first operational infectious disease forecast station, modeled after warning protocols developed in the meteorology community, was activated in 2011. The approach was originally pioneered in Haiti following the 2010 earthquake.
We assembled global event signature and forecast libraries that reflected locally diagnosed infectious disease activity and infrastructure impact in a rural community from a public health, veterinary, and human clinical medicine perspective. The deployment site is home to a variety of infectious disease including hantavirus, plague, tularemia, and West Nile in the context of high wildlife-livestock-human interfacing. Information derived from the issuance of forecasts coupled to situational awareness was shared with the public, local officials, public health officers, veterinarians, healthcare providers, and patients through various social media methods.
Provision of 30-60-90 day forecasts for routine and non-routine endemic infectious disease activity and impact facilitated better coordination of public health messaging and daily conversation with patients in the inpatient and outpatient settings. The signature of an unusual, infrastructure-disruptive outbreak of metapneumovirus and respiratory syncytial virus was recognized and communicated with enough time to activate effective clinical mitigation protocols. Cost estimates demonstrated financial benefit at a local level to anticipating surges of infectious disease activity with enough time to mitigate patient demand. Community-wide engagement with infectious disease forecasts and live event advisories included the promotion of proactive infection control and public health surveillance and response, healthcare provider recognition of non-routine infectious disease, clinical sampling and diagnostic testing protocols, clinician and patient education, and synchronization of proactive disease reporting both in the routine daily clinical setting and in times of crisis. Collateral benefit of consistent messaging delivered to the public by the participating entities was noted. Community awareness of the repertoire of indigenous infectious disease activity was expanded beyond the official public health notification list. Neither issuance of infectious disease forecasts nor advisories issued during crises triggered an influx of anxious well phone calls or visits to the medical system that was deemed operationally relevant.
Activation of a local infectious disease forecast station modeled after a local weather station promotes routine communication of a broader array of infectious disease activity than that monitored by public health; facilitates proactive, cost effective healthcare; and enabled recognition of unusual, disruptive infectious activity with enough time to enable mitigation of clinical, infrastructure, and financial impact to the community. Routine communication of comprehensive infectious disease forecast and situational awareness information promotes community adaptive fitness to a wide variety of infectious hazards. The results suggest it is possible to transform the traditional public health model of data collection and analysis to one of transparent and open data availability to support innovative reduction in morbidity and mortality.
PMCID: PMC3692870
biosurveillance; forecast; meteorology
21.  Hepatocellular carcinoma in a gene therapy research subject with ornithine transcarbamylase deficiency 
Molecular Genetics and Metabolism  2011;105(2):263-265.
A 66 year old woman who is a manifesting heterozygote for ornithine transcarbamylase deficiency (OTCD) presented with hepatocellular carcinoma (HCC). Fourteen years prior to this presentation she participated in a phase I gene therapy study which used an adenoviral vector, thought to be non-oncogenic, to deliver a normal OTC gene to hepatocytes [1]. A recent review of data collected through a national longitudinal study of individuals with urea cycle defects [2, 3] suggests that early urea cycle disorders (UCDs) are associated with hepatocellular damage and liver dysfunction in many cases. This may predispose an affected individual to a substantially increased risk of developing HCC, as has been observed in certain other inborn errors of metabolism. We speculate that the underlying urea cycle defect may be the cause of HCC in this individual.
PMCID: PMC3273986  PMID: 22129577
urea cycle disorders (UCDs); ornithine transcarbamylase deficiency (OTCD); hepatocellular carcinoma (HCC); gene therapy; adenovirus
22.  Analysis of Particle Content of Recombinant Adeno-Associated Virus Serotype 8 Vectors by Ion-Exchange Chromatography 
Human Gene Therapy Methods  2012;23(1):56-64.
Advances in adeno-associated virus (AAV)-mediated gene therapy have brought the possibility of commercial manufacturing of AAV vectors one step closer. To realize this prospect, a parallel effort with the goal of ever-increasing sophistication for AAV vector production technology and supporting assays will be required. Among the important release assays for a clinical gene therapy product, those monitoring potentially hazardous contaminants are most critical for patient safety. A prominent contaminant in many AAV vector preparations is vector particles lacking a genome, which can substantially increase the dose of AAV capsid proteins and lead to possible unwanted immunological consequences. Current methods to determine empty particle content suffer from inconsistency, are adversely affected by contaminants, or are not applicable to all serotypes. Here we describe the development of an ion-exchange chromatography-based assay that permits the rapid separation and relative quantification of AAV8 empty and full vector particles through the application of shallow gradients and a strong anion-exchange monolith chromatography medium.
Lock and colleagues describe the development of an ion-exchange (IEX) chromatography-based assay that permits the rapid separation and relative quantification of AAV8 empty and full vector particles. Unlike previously published particle assays, the IEX-based method described here requires little in the way of sample preparation or special reagents and eliminates interpretative counting and compounded errors.
PMCID: PMC4015067  PMID: 22428980
23.  Identification of the Galactose Binding Domain of the Adeno-Associated Virus Serotype 9 Capsid 
Journal of Virology  2012;86(13):7326-7333.
Adeno-associated virus serotype 9 (AAV9) vectors show promise for gene therapy of a variety of diseases due to their ability to transduce multiple tissues, including heart, skeletal muscle, and the alveolar epithelium of the lung. In addition, AAV9 is unique compared to other AAV serotypes in that it is capable of surpassing the blood-brain barrier and transducing neurons in the brain and spinal cord. It has recently been shown that AAV9 uses galactose as a receptor to transduce many different cell types in vitro, as well as cells of the mouse airway in vivo. In this study, we sought to identify the specific amino acids of the AAV9 capsid necessary for binding to galactose. By site-directed mutagenesis and cell binding assays, plus computational ligand docking studies, we discovered five amino acids, including N470, D271, N272, Y446, and W503, which are required for galactose binding that form a pocket at the base of the protrusions around the icosahedral 3-fold axes of symmetry. The importance of these amino acids for tissue tropism was also confirmed by in vivo studies in the mouse lung. Identifying the interactions necessary for AAV9 binding to galactose may lead to advances in vector engineering.
PMCID: PMC3416318  PMID: 22514350
24.  Humoral Immune Response to AAV 
Adeno-associated virus (AAV) is a member of the family Parvoviridae that has been widely used as a vector for gene therapy because of its safety profile, its ability to transduce both dividing and non-dividing cells, and its low immunogenicity. AAV has been detected in many different tissues of several animal species but has not been associated with any disease. As a result of natural infections, antibodies to AAV can be found in many animals including humans. It has been shown that pre-existing AAV antibodies can modulate the safety and efficacy of AAV vector-mediated gene therapy by blocking vector transduction or by redirecting distribution of AAV vectors to tissues other than the target organ. This review will summarize antibody responses against natural AAV infections, as well as AAV gene therapy vectors and their impact in the clinical development of AAV vectors for gene therapy. We will also review and discuss the various methods used for AAV antibody detection and strategies to overcome neutralizing antibodies in AAV-mediated gene therapy.
PMCID: PMC3799231  PMID: 24151496
adeno-associated virus; AAV; neutralizing antibody; immune response; gene therapy
25.  Challenges in using 18 F-fluorodeoxyglucose-PET-CT to define a biological radiotherapy boost volume in locally advanced pancreatic cancer 
The best method of identifying regions within pancreatic tumours that might benefit from an increased radiotherapy dose is not known. We investigated the utility of pre-treatment FDG-PET in predicting the spatial distribution of residual metabolic activity following chemoradiotherapy (CRT) in locally advanced pancreatic cancer (LAPC).
17 patients had FDG-PET/CT scans at baseline and six weeks post-CRT. Tumour segmentation was performed at 40% and 50% of SUVmax at baseline and 60%, 70%, 80% and 90% post-CRT. FDG-PET scans were non-rigidly registered to the radiotherapy planning CT using the CT component of the FDG-PET/CT. Percentage overlap of the post-CRT volumes with the pre-CRT volumes with one another and the gross tumour volume (GTV) was calculated.
SUVmax decreased during CRT (median pre- 8.0 and post- 3.6, p < 0.0001). For spatial correlation analysis, 9 pairs of scans were included (Four were excluded following complete metabolic response, one patient had a non-FDG avid tumour, one had no post-CRT imaging, one had diffuse FDG uptake that could not be separated from normal tissues and one had an elevated blood glucose). The Pre40% and 50% of SUVmax volumes covered a mean of 50.8% and 30.3% of the GTV respectively. The mean% overlap of the 90%, 80%, 70%, 60% of SUVmax post-CRT with the Pre40% and Pre50% volumes were 83.3%, 84.0%, 83.7%, 77.9% and 77.8%, 69.9%, 74.5%, 64.8% respectively.
Regions of residual metabolic activity following CRT can be predicted from the baseline FDG-PET and could aid definition of a biological target volume for non-uniform dose prescriptions.
PMCID: PMC4078370  PMID: 24962658
Pancreatic cancer; PET-CT; Residual metabolic activity; Intra-tumour heterogeneity; Biological target volume

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