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1.  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
2.  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
3.  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
4.  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
5.  Chromosome 9p21 Single Nucleotide Polymorphisms are Not Associated with Recurrent Myocardial Infarction in Patients with Established Coronary Artery Disease 
Chromosome 9p21 single-nucleotide-polymorphisms (SNPs) have shown to be associated with coronary heart disease in multiple studies. We aimed to identify whether these SNPs are associated with recurrent myocardial infarction (MI), revascularization, or death in acute-coronary-syndromes (ACS) patients or those undergoing coronary artery bypass grafting (CABG).
Methods and Results
TexGen registry participants with ACS (n=2,067) or CABG (n=1,176) were evaluated. We assessed whether 9p21 SNPs (rs1333049, rs2383206, rs10757278, rs10757274) were associated with recurrent MI (primary outcome), recurrent revascularization, or death (secondary outcomes) at ≈ 3.2 years of follow-up. Carriers of risk allele (C) for rs1333049 presented at an earlier age (62 vs. 63.5 years in non-carriers, p=0.0004) with more extensive disease (number of vessels with significant stenosis =1.9 vs. 1.7 in non-carriers, p=0.001) in the ACS group. In adjusted models, C allele was not associated with recurrent MI (HR 1.01, 95%CI 0.74–1.38), recurrent revascularization (HR 0.98, 95%CI 0.78–1.23), or death (HR 0.91, 95%CI 0.69–1.18) in ACS or CABG group (HR 0.64, 95%CI 0.40–1.05 for recurrent MI; HR 0.98, 95%CI 0.61–1.55 for recurrent revascularization; and HR 0.89, 95%CI 0.61–1.30 for death). Results were similar for the other 3 SNPs.
9p21 SNPs were not associated with recurrent MI, revascularization, or mortality after ACS or CABG. However, individuals with the rs1333049 C allele may present with earlier and more extensive disease.
PMCID: PMC3837386  PMID: 22322877
9p21 locus; recurrent myocardial infarction; recurrent revascularization; mortality
6.  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
7.  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
8.  Impact of Pre-Existing Immunity on Gene Transfer to Nonhuman Primate Liver with Adeno-Associated Virus 8 Vectors 
Human Gene Therapy  2011;22(11):1389-1401.
Vectors based on the primate-derived adeno-associated virus serotype 8 (AAV8) are being evaluated in preclinical and clinical models. Natural infections with related AAVs activate memory B cells that produce antibodies capable of modulating the efficacy and safety of the vector. We have evaluated the biology of AAV8 gene transfer in macaque liver, with a focus on assessing the impact of pre-existing humoral immunity. Twenty-one macaques with various levels of AAV neutralizing antibody (NAb) were injected intravenously with AAV8 vector expressing green fluorescent protein. Pre-existing antibody titers in excess of 1:10 substantially diminished hepatocyte transduction that, in the absence of NAbs, was highly efficient. Vector-specific NAb diminished liver deposition of genomes and unexpectedly increased genome distribution to the spleen. The majority of animals showed high-level and stable sequestration of vector capsid protein by follicular dendritic cells of splenic germinal centers. These studies illustrate how natural immunity to a virus that is related to a vector can impact the efficacy and potential safety of in vivo gene therapy. We propose to use the in vitro transduction inhibition assay to evaluate research subjects before gene therapy and to preclude from systemic AAV8 trials those that have titers in excess of 1:10.
Wang and colleagues evaluate the impact of preexisting humoral immunity on adeno-associated virus 8 (AAV8)-mediated gene transfer to macaque livers. They injected AAV8 vectors expressing green fluorescent protein into 21 macaques with various levels of AAV-neutralizing antibody (Nab), and found that NAb titers above 1:10 significantly impaired transduction and affected distribution of vector genomes.
PMCID: PMC3225046  PMID: 21476868
9.  Correction: AAV9 Targets Cone Photoreceptors in the Nonhuman Primate Retina 
PLoS ONE  2013;8(8):10.1371/annotation/64b90996-4634-4c63-b737-634397b0b445.
PMCID: PMC3738648
10.  Pseudotyped Adeno-associated Viral Vector Tropism and Transduction Efficiencies in Murine Wound Healing 
Cell specific gene transfer and sustained transgene expression are goals of cutaneous gene therapy for tissue repair and regeneration. Adeno-associated virus serotype 2 (AAV2/2) mediated gene transfer to the skin results in stable transgene expression in the muscle fascicles of the panniculus carnosus in mice, with minimal gene transfer to the dermal or epidermal elements. We hypothesized that pseudotyped AAV vectors may have a unique and characteristic tropism and transduction efficiency profile for specific cells in the cutaneous wounds. We compared transduction efficiencies of cells in the epidermis, cells in the dermis, and the fascicles of the panniculus carnosus by AAV2/2 and three pseudotyped AAV vectors, AAV2/5, AAV2/7 and AAV2/8 in a murine excisional wound model. AAV2/5 and AAV2/8 result in significantly enhanced transduction of cells both in the epidermis and the dermis compared to AAV2/2. AAV2/5 transduces both the basilar and supra-basilar keratinocytes. In contrast, AAV2/8 transduces mainly supra-basilar keratinocytes. Both AAV2/7 and AAV2/8 result in more efficient gene transfer to the muscular panniculus carnosus compared to AAV2/2. The capsid of the different pseudotyped AAV vectors produces distinct tropism and efficiency profiles in the murine wound healing model. Both AAV2/5 and AAV2/8 administration result in significantly enhanced gene transfer. To further characterize cell specific transduction and tropism profiles of the AAV pseudotyed vectors, we performed in vitro experiments using human and mouse primary dermal fibroblasts. Our data demonstrates that pseudotyping strategy confers a differential transduction of dermal fibroblasts, with higher transduction of both human and murine cells by AAV2/5 and AAV2/8 at early and later time points. At later time points, AAV2/2 demonstrates increased transduction. Interestingly, AAV2/8 appears to be more efficacious in transducing human cells as compared to AAV2/5. The pseudotype-specific pattern of transduction and tropism observed both in vivo and in vitro suggests that choice of AAV vectors should be based on the desired target cell and the timing of transgene expression in wound healing for gene transfer therapy in dermal wounds.
PMCID: PMC3654388  PMID: 22713157
Gene Therapy; Wound Healing; Adeno-associated Virus; Pseudotyped Vectors
11.  Inflammation Causes Loss of Adeno-Associated Virus-Mediated Transgene Expression in Mouse Liver 
Gastroenterology  2011;141(1):348-357.e3.
Background & Aims
Transgenes delivered to livers of mice via adeno-associated virus (AAV) are expressed stably, via induction of immune tolerance. However, transgene expression is lost in higher-order primates. We investigated whether inflammatory processes, which likely differ between species, affect the stability of transgene expression.
We developed a mouse model of vector-unrelated, systemic inflammation following AAV-mediated transfer of genes to liver.
Inflammation eliminated previously stable expression of trangenes delivered by AAV; the limited tissue destruction and persistence of AAV genomes implicated immune responses that were not mediated by cytotoxic T cells. Tumor necrosis factor (TNF)-α downregulated transgene expression from AAV, indicating a role for inflammatory cytokines in loss of transgene expression.
Inflammation and inflammatory cytokines such as TNF-α can reduce AAV-mediated expression of transgenes in livers of mice. Inflammation might therefore affect expression of transgenes from viral vectors in humans.
PMCID: PMC3269906  PMID: 21640112
Hepatic gene therapy; loss of tolerance; immune response; liver inflammation
12.  Adenoviruses in Fecal Samples from Asymptomatic Rhesus Macaques, United States 
Emerging Infectious Diseases  2012;18(7):1081-1088.
Isolates contained fiber genes similar to those of adenovirus strains that cause infectious diarrhea in humans.
Adenoviruses can cause infectious diarrheal disease or respiratory infections in humans; 2 recent reports have indicated probable human infection with simian adenoviruses (SAdVs). To assess the possibility of animal-to-human transmission of SAdVs, we tested fecal samples from asymptomatic rhesus macaques housed in 5 primate facilities in the United States and cultured 23 SAdV isolates. Of these, 9 were purified and completely sequenced; 3 SAdV samples from the American Type Culture Collection (SAdV-6, SAdV-18, and SAdV-20) were also completely sequenced. The sequence of SAdV-18 was closely related to that of human adenovirus F across the whole genome, and the new isolates were found to harbor 2 fiber genes similar to those of human adenovirus (HAdV) strains HAdV-40 and HAdV-41, which can cause infectious diarrhea. The high prevalence of adenoviruses in fecal samples from asymptomatic rhesus macaques and the similarity of the isolates to human strains indicates the possibility of animal-to-human transmission of SAdVs.
PMCID: PMC3376797  PMID: 22709783
adenovirus; rhesus; macaques; fiber; hexon; DNA; polymerase; United States; feces; stool; fecal samples; viruses; asymptomatic; subclinical infection; PCR; monkeys
13.  Genome-wide DNA methylation analysis reveals novel epigenetic changes in chronic lymphocytic leukemia 
Epigenetics  2012;7(6):567-578.
We conducted a genome-wide DNA methylation analysis in CD19+ B-cells from chronic lymphocytic leukemia (CLL) patients and normal control samples using reduced representation bisulfite sequencing (RRBS). The methylation status of 1.8–2.3 million CpGs in the CLL genome was determined; about 45% of these CpGs were located in more than 23,000 CpG islands (CGIs). While global CpG methylation was similar between CLL and normal B-cells, 1764 gene promoters were identified as being differentially methylated in at least one CLL sample when compared with normal B-cell samples. Nineteen percent of the differentially methylated genes were involved in transcriptional regulation. Aberrant hypermethylation was found in all HOX gene clusters and a significant number of WNT signaling pathway genes. Hypomethylation occurred more frequently in the gene body including introns, exons, and 3′-UTRs in CLL. The NFATc1 P2 promoter and first intron was found to be hypomethylated and correlated with upregulation of both NFATc1 RNA and protein expression levels in CLL suggesting that an epigenetic mechanism is involved in the constitutive activation of NFAT activity in CLL cells. This comprehensive DNA methylation analysis will further our understanding of the epigenetic contribution to cellular dysfunction in CLL.
PMCID: PMC3398986  PMID: 22534504
DNA Methylation; NFATc1; chronic lymphocytic leukemia; next-generation sequencing; reduced representation bisulfite sequencing
14.  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
15.  Lung Homing CTLs and Their Proliferation Ability are Important Correlates of Vaccine Protection against Influenza 
Vaccine  2010;28(35):5669-5675.
We conducted a study to evaluate the protective efficacy in mice of vaccination with novel adenovirus vectors expressing an influenza A nucleoprotein (AdFluA-NP) based on isolates from non-human primates. In a previous study, we had observed that AdFluA-NP vectors can induce similar T cell responses in mice yet differ in ability to protect animals from lethal challenge with influenza A virus. To better define correlates of protection, we extended our study design to include additional novel AdFluA-NP vectors, and to evaluate cytotoxic T lymphocyte (CTL) responses in the spleens and lungs of immunized mice prior to virus challenge. As in our previous study, all vectors induced similar numbers of antigen-specific interferon gamma (IFN-γ) secreting T cells and memory T cells in the spleen four weeks post-immunization, but differed in their ability to protect the animals from lethal infection. However, cytokine-secreting NP antigen-specific CTLs in the lungs of mice from immunization groups that survived lethal challenge showed greater proliferative ability and higher CD27 expression. In addition, NP antigen-specific peripheral blood lymphocytes from protected mice showed greater proliferative ability after ex vivo stimulation. Our results provide additional correlates of protection that should be considered when developing anti-influenza vaccines.
PMCID: PMC3269910  PMID: 20600493
Influenza; vaccine; CTL
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.  Gene Therapy for Rare Diseases: Summary of a National Institutes of Health Workshop, September 13, 2012 
Human Gene Therapy  2013;24(4):355-362.
Gene therapy has shown clinical efficacy for several rare diseases, using different approaches and vectors. The Gene Therapy for Rare Diseases workshop, sponsored by the National Institutes of Health (NIH) Office of Biotechnology Activities and Office of Rare Diseases Research, brought together investigators from different disciplines to discuss the challenges and opportunities for advancing the field including means for enhancing data sharing for preclinical and clinical studies, development and utilization of available NIH resources, and interactions with the U.S. Food and Drug Administration.
PMCID: PMC3631014  PMID: 23517518
18.  Efficient Serotype-Dependent Release of Functional Vector into the Culture Medium During Adeno-Associated Virus Manufacturing 
Human Gene Therapy  2010;21(10):1251-1257.
Vandenberghe and colleagues demonstrate that in contrast to AAV2, most other AAV serotypes are primarily released into the media of calcium phosphate-transfected production cultures and not retained in the cell lysate. Based on this observation, the authors develop a scalable method for isolating functional vector particles from culture medium. This finding should help to streamline the production process for both laboratory and clinical applications of AAV.
Vectors based on adeno-associated virus (AAV) are the subject of increasing interest as research tools and agents for in vivo gene therapy. A current limitation on the technology is the versatile and scalable manufacturing of vector. On the basis of experience with AAV2-based vectors, which remain strongly cell associated, AAV vector particles are commonly harvested from cell lysates, and must be extensively purified for use. We report here that vectors based on other AAV serotypes, including AAV1, AAV8, and AAV9, are found in abundance in, and can be harvested from, the medium of production cultures carried out with or without serum. For AAV2, this difference in compartmentalization is largely due to the affinity of the AAV2 particle for heparin, because an AAV2 variant in which the heparin-binding motif has been ablated gives higher yields and is efficiently released from cells. Vector particles isolated from the culture medium appear to be functionally equivalent to those purified from cell lysates in terms of transduction efficiency in vitro and in vivo, immunogenicity, and tissue tropism. Our findings will directly lead to methods for increasing vector yields and simplifying production processes for AAV vectors, which should facilitate laboratory-scale preparation and large-scale manufacture.
PMCID: PMC2957237  PMID: 20649475
19.  A systematic evaluation of miRNA:mRNA interactions involved in the migration and invasion of breast cancer cells 
In this study we performed a systematic evaluation of functional miRNA-mRNA interactions associated with the invasiveness of breast cancer cells using a combination of integrated miRNA and mRNA expression profiling, bioinformatics prediction, and functional assays. Analysis of the miRNA expression identified 11 miRNAs that were differentially expressed, including 7 down-regulated (miR-200c, miR-205, miR-203, miR-141, miR-34a, miR-183, and miR-375) and 4 up-regulated miRNAs (miR-146a, miR-138, miR-125b1 and miR-100), in invasive cell lines when compared to normal and less invasive cell lines. Transfection of miR-200c, miR-205, and miR-375 mimics into MDA-MB-231 cells led to the inhibition of in vitro cell migration and invasion. The integrated analysis of miRNA and mRNA expression identified 35 known and novel target genes of miR-200c, miR-205, and mir-375, including CFL2, LAMC1, TIMP2, ZEB1, CDH11, PRKCA, PTPRJ, PTPRM, LDHB, and SEC23A. Surprisingly, the majority of these genes (27 genes) were target genes of miR-200c, suggesting that miR-200c plays a pivotal role in regulating the invasiveness of breast cancer cells. We characterized one of the target genes of miR-200c, CFL2, and demonstrated that CFL2 is overexpressed in aggressive breast cancer cell lines and can be significantly down-regulated by exogenous miR-200c. Tissue microarray analysis further revealed that CFL2 expression in primary breast cancer tissue correlated with tumor grade. The results obtained from this study may improve our understanding of the role of these candidate miRNAs and their target genes in relation to breast cancer invasiveness and ultimately lead to the identification of novel biomarkers associated with prognosis.
PMCID: PMC3599769  PMID: 23497265
Breast cancer; MicroRNA target genes; Migration; Invasion
20.  Impact of Diabetes Among Revascularized Patients in Japan and the U.S. 
Diabetes Care  2012;35(3):654-659.
Approximately 25% of patients who undergo percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) have diabetes, and the diagnosis of diabetes roughly doubles the mortality risk associated with coronary artery disease. However, the impact of diabetes may differ according to ethnicity. Our objective was to examine the impact of diabetes on long-term survival among U.S. and Japanese patients who underwent PCI or CABG.
For the current analysis, we included 8,871 patients from a Japanese multicenter registry (Coronary Revascularization Demonstrating Outcome database in Kyoto; median follow-up 3.5 years; interquartile range [IQR] 2.6–4.3) and 7,229 patients from a U.S. multipractice registry (Texas Heart Institute Research Database; median follow-up 5.2 years; IQR 3.8–6.5).
Diabetes was more prevalent among Japanese than U.S. patients (39.2 vs. 31.0%; P < 0.001). However, after revascularization, long-term all-cause mortality was lower in diabetic Japanese patients than in diabetic U.S. patients (85.4 vs. 82.2%; log-rank test P = 0.009), whereas it was similar in nondiabetic Japanese and U.S. patients (89.1 vs. 89.5%; P = 0.50). The national difference in crude mortality was also significant among insulin-using patients with diabetes (80.8 vs. 74.9%; P = 0.023). When long-term mortality was adjusted for known predictors, U.S. location was associated with greater long-term mortality risk than Japanese location among nondiabetic patients (hazard ratio 1.58 [95% CI 1.32–1.88]; P < 0.001) and, especially, diabetic patients (1.88 [1.54–2.30]; P < 0.001).
Although diabetes was less prevalent in U.S. patients than in Japanese patients, U.S. patients had higher overall long-term mortality risk. This difference was more pronounced in diabetic patients.
PMCID: PMC3322729  PMID: 22301120
21.  Preclinical Evaluation of a Clinical Candidate AAV8 Vector for Ornithine Transcarbamylase (OTC) Deficiency Reveals Functional Enzyme from Each Persisting Vector Genome 
Molecular Genetics and Metabolism  2011;105(2):203-211.
Ornithine transcarbamylase deficiency (OTCD), the most common and severe urea cycle disorder, is an excellent model for developing liver-directed gene therapy. No curative therapy exists except for liver transplantation which is limited by available donors and carries significant risk of mortality and morbidity. Adeno-associated virus 8 (AAV8) has been shown to be the most efficient vector for liver-directed gene transfer and is currently being evaluated in a clinical trial for treating hemophilia B. In this study, we generated a clinical candidate vector for a proposed OTC gene therapy trial in humans based on a self-complementary AAV8 vector expressing codon-optimized human OTC (hOTCco) under the control of a liver-specific promoter. Codon-optimization dramatically improved the efficacy of OTC gene therapy. Supraphysiological expression levels and activity of hOTC were achieved in adult spfash mice following a single intravenous injection of hOTCco vector. Vector doses as low as 1×1010 genome copies (GC) achieved robust and sustained correction of the OTCD biomarker orotic aciduria and clinical protection against an ammonia challenge. Functional expression of hOTC in 40% of liver areas was found in mice treated with a low vector dose of 1×109 GC. We suggest that the clinical candidate vector we have developed has the potential to achieve therapeutic effects in OTCD patients.
PMCID: PMC3270700  PMID: 22133298
Adeno-associated viruses (AAV); liver gene therapy; ornithine transcarbamylase deficiency (OTCD); codon optimization
22.  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
23.  RPGR-Associated Retinal Degeneration in Human X-Linked RP and a Murine Model 
We investigated the retinal disease due to mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene in human patients and in an Rpgr conditional knockout (cko) mouse model.
XLRP patients with RPGR-ORF15 mutations (n = 35, ages at first visit 5–72 years) had clinical examinations, and rod and cone perimetry. Rpgr-cko mice, in which the proximal promoter and first exon were deleted ubiquitously, were back-crossed onto a BALB/c background, and studied with optical coherence tomography and electroretinography (ERG). Retinal histopathology was performed on a subset.
Different patterns of rod and cone dysfunction were present in patients. Frequently, there were midperipheral losses with residual rod and cone function in central and peripheral retina. Longitudinal data indicated that central rod loss preceded peripheral rod losses. Central cone-only vision with no peripheral function was a late stage. Less commonly, patients had central rod and cone dysfunction, but preserved, albeit abnormal, midperipheral rod and cone vision. Rpgr-cko mice had progressive retinal degeneration detectable in the first months of life. ERGs indicated relatively equal rod and cone disease. At late stages, there was greater inferior versus superior retinal degeneration.
RPGR mutations lead to progressive loss of rod and cone vision, but show different patterns of residual photoreceptor disease expression. Knowledge of the patterns should guide treatment strategies. Rpgr-cko mice had onset of degeneration at relatively young ages and progressive photoreceptor disease. The natural history in this model will permit preclinical proof-of-concept studies to be designed and such studies should advance progress toward human therapy.
Progress in treating canine RPGR disease prompted us to characterize patients with RPGR-ORF15 mutations and provide a detailed natural history of a novel Rpgr-mutant mouse for further proof-of-concept experiments.
PMCID: PMC3422104  PMID: 22807293
24.  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
25.  Predicting Early Death after Cardiovascular Surgery by Using the Texas Heart Institute Risk Scoring Technique (THIRST) 
Texas Heart Institute Journal  2013;40(2):156-162.
Preoperative risk-prediction models are an important tool in contemporary surgical practice. We developed a risk-scoring technique for predicting in-hospital death for cardiovascular surgery patients. From our institutional database, we obtained data on 21,120 patients admitted from 1995 through 2007. The outcome of interest was early death (in-hospital or within 30 days of surgery). To identify mortality predictors, multivariate logistic regression was performed on data from 14,030 patients from 1995 through 2002 and risk scores were computed to stratify patients (low-, medium-, and high-risk). A recalibrated model was then created from the original risk scores and validated on data from 7,090 patients from 2003 through 2007. Significant predictors of death included urgent surgery within 48 hours of admission, advanced age, renal insufficiency, repeat coronary artery bypass grafting, repeat aortic aneurysm repair, concomitant aortic aneurysm or left ventricular aneurysm repair with coronary bypass or valvular surgery, and preoperative intra-aortic balloon pump support. Because the original model overpredicted death for operations performed from 2003 through 2007, this was adjusted for by applying the recalibrated model. Applying the recalibrated model to the validation set revealed predicted mortality rates of 1.7%, 4.2%, and 13.4% and observed rates of 1.1%, 5.1%, and 13%, respectively. Because our model discriminates risk groups by using preoperative clinical criteria alone, it can be a useful bedside tool for identifying patients at greater risk of early death after cardiovascular surgery, thereby facilitating clinical decision-making. The model can be recalibrated for use in other types of patient populations.
PMCID: PMC3649797  PMID: 23678213
Cardiac surgical procedures/mortality; evaluation studies as topic; hospital mortality; logistic models; models, statistical; outcome assessment (health care)/methods; predictive value of tests; regression analysis of tests; risk assessment/classification/methods/statistics & numerical data; risk factors; ROC curve; statistics as topic; United States/epidemiology

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