Andrea Amalfitano

Loss-of-function mutations in FGFR1 cause autosomal dominant Kallmann syndrome

We took advantage of overlapping interstitial deletions at chromosome 8p11–p12 in two individuals with contiguous gene syndromes and defined an interval of roughly 540 kb associated with a dominant form of Kallmann syndrome, KAL2. We establish here that loss-of-function mutations in FGFR1 underlie KAL2 whereas a gain-of-function mutation in FGFR1 has been shown to cause a form of craniosynostosis. Moreover, we suggest that the KAL1 gene product, the extracellular matrix protein anosmin-1, is involved in FGF signaling and propose that the gender difference in anosmin-1 dosage (because KAL1 partially escapes X inactivation) explains the higher prevalence of the disease in males.

TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells.

Mechanical deflection of the sensory hair bundles of receptor cells in the inner ear causes ion channels located at the tips of the bundle to open, thereby initiating the perception of sound. Although some protein constituents of the transduction apparatus are known, the mechanically gated transduction channels have not been identified in higher vertebrates. Here, we investigate TRP (transient receptor potential) ion channels as candidates and find one, TRPA1 (also known as ANKTM1), that meets criteria for the transduction channel. The appearance of TRPA1 messenger RNA expression in hair cell epithelia coincides developmentally with the onset of mechanosensitivity. Antibodies to TRPA1 label hair bundles, especially at their tips, and tip labelling disappears when the transduction apparatus is chemically disrupted. Inhibition of TRPA1 protein expression in zebrafish and mouse inner ears inhibits receptor cell function, as assessed with electrical recording and with accumulation of a channel-permeant fluorescent dye. TRPA1 is probably a component of the transduction channel itself.

Adenovirus Vector-Induced Innate Inflammatory Mediators, MAPK Signaling, As Well As Adaptive Immune Responses Are Dependent upon Both TLR2 and TLR9 In Vivo

Adenovirus (Ad) vectors are promising candidates for both gene transfer and vaccine applications. In this study, we investigated the role of TLR2 in innate and adaptive immune responses to Ad and/or the transgene it expresses following systemic injection. We found that Ad directly activates ERK1/2 in vivo, but that initiation of ERK1/2 activation is primarily a MyD88/TLR2-independent, but Kupffer cell-dependent, event. The complexity of Ad-induced innate immune responses was confirmed when we also found that both TLR2 and MyD88 functions are required for the sustained activation of ERK1/2. Although we found that the initial activation of NF-κB by Ads is dependent upon MyD88, but independent of TLR2 in (non-Kupffer cells) the liver, TLR2 significantly influenced the Ad-induced late phase NF-κB activation. These very rapid responses were positively correlated with subsequent innate immune responses to the Ad vector, as our results confirmed that the induction of several cytokines and chemokines, and the expression of innate immune response genes following Ad injection were TLR2 dependent in vivo. The requirement of TLR2 in Ad-induced innate responses also correlated with significantly altered adaptive immune responses. For example, our results demonstrate that the generation of Ad-neutralizing Abs, and anti-transgene-specific Abs elicited subsequent to Ad vector treatments, are both dependent upon TLR2 functionality. Finally, we found that several Ad-induced innate immune responses are dependent on both TLR2 and TLR9. Therefore, this study confirms that several (but not all) Ad-induced innate and adaptive immune responses are TLR dependent.

Adenovirus Infection Triggers a Rapid, MyD88-Regulated Transcriptome Response Critical to Acute-Phase and Adaptive Immune Responses In Vivo

ABSTRACT Nearly 50 years ago, the discovery of interferon prompted the notion that host cells innately respond to viral invasion. Since that time, technological advances have allowed this response to be extensively characterized and dissected in vitro. However, these advances have only recently been applied to highly complex, in vivo biological systems. To this end, we exploited high-titer adenovirus (Ad) vectors to globally investigate the innate immune response to nonenveloped viral infection in vivo. Our results indicated a potent cellular transcriptome response shortly after infection, with global assessments revealing significant dysregulation in ∼15% of the measured transcripts derived from Ad vector-transduced tissue. Bioinformatics-based transcriptome analysis revealed a complex innate response to Ad infection, with induction of proinflammatory responses (and suppression of metabolism and mitochondrial genes) akin to those observed when mice are challenged with lipopolysaccharide. Despite this commonality, there were many unique aspects of the Ad-dependent transcriptome response, including the upregulation of several RNA regulatory mechanisms and apoptosis-related pathways, accompanied by the suppression of lysosomal and endocytic genes. Our results also implicated the Toll-like receptors (TLRs) in these responses, prompting specific investigations into this pathway. By using MyD88KO mice, our results confirmed that Ad-induced dysregulation of five functionally related gene clusters are significantly dependent on this TLR adaptor gene. MyD88 deficiency also resulted in significantly diminished, although not abolished, adaptive and acute-phase immune responses to Ad, confirming the transcriptome data, as well as specifically identifying MyD88 as a significant Ad immunity amplifier and regulator in vivo.

Isolation and characterization of packaging cell lines that coexpress the adenovirus E1, DNA polymerase, and preterminal proteins: implications for gene therapy

Current generation adenovirus (Ad) vectors are deleted for the E1 region of genes and require propagation in E1 expressing 293 cells. Expression of genes delivered by Ad vectors into immunocompetent hosts is generally transient since the current vectors are not completely replication defective. Viral proteins expressed by Ad vectors, in part, induce a rapid, T cell-mediated loss of the transduced cells. Introduction of temperature-sensitive point mutations into new Ad vectors may be of limited usefulness in prolonging transduced gene expression in vivo. Isolation of new Ad vectors deleted for genes required for normal Ad growth may further prevent Ad protein expression. These new vectors will need to be grown in 293 cells capable of coexpressing other Ad genes. Unfortunately, many of the Ad genes are toxic when coexpressed in 293 cells. We describe the isolation of E1 expressing 293 cells which also express both the Ad polymerase and preterminal proteins, both of which are essential to normal Ad growth. The isolation of new Ad vectors deleted for the E1, polymerase and preterminal proteins are predicted to have many advantageous properties, including the prolongation of transduced foreign gene expression in vivo.

Cutting Edge: Coding Single Nucleotide Polymorphisms of Endoplasmic Reticulum Aminopeptidase 1 Can Affect Antigenic Peptide Generation In Vitro by Influencing Basic Enzymatic Properties of the Enzyme

ER aminopeptidase 1 (ERAP1) customizes antigenic peptide precursors for MHC class I presentation and edits the antigenic peptide repertoire. Coding single nucleotide polymorphisms (SNPs) in ERAP1 were recently linked with predisposition to autoimmune disease, suggesting a link between pathogenesis of autoimmunity and ERAP1-mediated Ag processing. To investigate this possibility, we analyzed the effect that disease-linked SNPs have on Ag processing by ERAP1 in vitro. Michaelis–Menten analysis revealed that the presence of SNPs affects the Michaelis constant and turnover number of the enzyme. Strikingly, specific ERAP1 allele-substrate combinations deviate from standard Michaelis–Menten behavior, demonstrating substrate-inhibition kinetics; to our knowledge, this phenomenon has not been described for this enzyme. Cell-based Ag-presentation analysis was consistent with changes in the substrate inhibition constant Ki, further supporting that ERAP1 allelic composition may affect Ag processing in vivo. We propose that these phenomena should be taken into account when evaluating the possible link between Ag processing and autoimmunity.

Transient Pretreatment With Glucocorticoid Ablates Innate Toxicity of Systemically Delivered Adenoviral Vectors Without Reducing Efficacy

More than 300 human clinical trials utilize recombinant adenoviruses (rAds) as a gene transfer vector, confirming that rAds continue to be of high clinical interest. A primary weakness of rAds is their known propensity to trigger an innate, proinflammatory immune response rapidly after high-dose, systemic administration. In this study, we investigated what affects that pre-emptive treatment with anti-inflammatory glucocorticoids might have upon Ad vector-triggered inflammatory immune responses. We found that a simple pretreatment regimen with Dexamethasone (DEX) can significantly reduce most Ad-induced innate immune responses. DEX prevented rAd induction of systemic cytokine/chemokine releases in a dose-dependent fashion, with higher dosages preventing rAd induction of acute thrombocytopenia, endothelial cell activation, proinflammatory gene induction, and leukocyte infiltration into transduced organs. Transient glucocorticoid pretreatment also significantly reduced rAd-induced adaptive immune responses, including a decreased induction of Ad-neutralizing antibodies (NAbs). Importantly, use of DEX did not reduce the efficacy of rAd-mediated gene transduction nor rAd-derived transgene expression. Our results demonstrate that a simple, pre-emptive and transient glucocorticoid pretreatment is a viable approach to reduce rAd-associated acute toxicities that currently limit the use of Ad vectors in systemic clinical applications.

Nephrotic Syndrome Complicating α-Glucosidase Replacement Therapy for Pompe Disease

We report a patient with Pompe disease who developed reversible nephrotic syndrome during prolonged, high-dose, experimental, enzyme replacement therapy with recombinant human acid α-glucosidase (rhGAA). Because of the development of antibodies to rhGAA and concomitant clinical decline, escalating doses of rhGAA were administered as part of an experimental immune tolerance regimen. Histologic evaluation of kidney tissue revealed glomerular deposition of immune complexes containing rhGAA itself, in a pattern of membranous nephropathy. To our knowledge, this is the first reported case of nephrotic syndrome occurring during enzyme replacement therapy. The nephrotic syndrome gradually resolved after the rhGAA dose was decreased, indicating that decreasing the antigenic load can ameliorate glomerular immune complex deposition associated with enzyme replacement in a highly sensitized patient.

Liver Toxicities Typically Induced by First-Generation Adenoviral Vectors Can Be Reduced by Use of E1, E2b-Deleted Adenoviral Vectors

Adenoviral vectors from which the E1 region has been deleted ([E1(-)] Ad) are known to induce strong immune responses after systemic delivery. In this study we have evaluated liver toxicities in mice after intravenous injection with high doses of [E1(-)] or modified [E1(-), E2b(-)] Ad vectors (both expressing the bacterial beta-galactosidase [lacZ] marker gene) in C57BL/6, BALB/c, and SCID mice. Our data demonstrate a marked reduction in maximal liver toxicities and pathologies (typically noted at 21 days postinjection) with the use of the [E1(-), E2b(-)] modified vector in all strains of mice tested. Our data also demonstrated that despite the use of the [E1(-), E2b(-)] Ad vector, significant liver toxicities were still observed. To address this issue and the fact that the lacZ gene was perceived as a foreign antigen in the immune-competent C57BL/6 and BALB/c mice, we similarly injected mice tolerant of LacZ (lacZ-TG). In contrast to our studies in C57BL/6 and BALB/c mice, LacZ-TG mice exhibited virtually no evidence of hepatotoxicity after intravenous injection with the [E1(-), E2b(-)] vector, in contrast to use of the [E1(-)] Ad vector. Our results demonstrate that the [E1(-), E2b(-)] Ad vector class can reduce liver toxicities typically ascribed to Ad vector-mediated gene transfer after transfer of a highly immunogenic or foreign gene, whereas transfer of a transgene that is perceived as nonforeign by the host can be delivered with virtually no evidence of toxicity. On the basis of a careful review of the literature, these improvements in vector safety rival those noted with other, more significantly modified Ad vectors described to date.

Overcoming pre-existing adenovirus immunity by genetic engineering of adenovirus-based vectors

Adenovirus (Ad)-based vectors offer several benefits showing their potential for use in a variety of vaccine applications. Recombinant Ad-based vaccines possess potent immunogenic potential, capable of generating humoral and cellular immune responses to a variety of pathogen-specific antigens expressed by the vectors. Ad5 vectors can be readily produced, allowing for usage in thousands of clinical trial subjects. This is now coupled with a history of safe clinical use in the vaccine setting. However, traditional Ad5-based vaccines may not be generating optimal antigen-specific immune responses, and generate diminished antigen-specific immune responses when pre-existing Ad5 immunity is present. These limitations have driven initiation of several approaches to improve the efficacy of Ad-based vaccines, and/or allow modified vaccines to overcome pre-existing Ad immunity. These include: generation of chemically modified Ad5 capsids; generation of chimeric Ads; complete replacement of Ad5-based vaccine platforms with alternative (human and non-human origin) Ad serotypes, and Ad5 genome modification approaches that attempt to retain the native Ad5 capsid, while simultaneously improving the efficacy of the platform as well as minimizing the effect of pre-existing Ad immunity. Here we discuss recent advances in- and limitations of each of these approaches, relative to their abilities to overcome pre-existing Ad immunity.