Sadik H. Kassim

In Vivo Ablation of CD11c-Positive Dendritic Cells Increases Susceptibility to Herpes Simplex Virus Type 1 Infection and Diminishes NK and T-Cell Responses

ABSTRACT The precise role of each of the seven individual CD11c+ dendritic cell subsets (DCs) identified to date in the response to viral infections is not known. DCs serve as critical links between the innate and adaptive immune responses against many pathogens, including herpes simplex virus type 1 (HSV-1). The role of DCs as mediators of resistance to HSV-1 infection was investigated using CD11c-diphtheria toxin (DT) receptor-green fluorescent protein transgenic mice, in which DCs can be transiently depleted in vivo by treatment with low doses of DT. We show that ablation of DCs led to enhanced susceptibility to HSV-1 infection in the highly resistant C57BL/6 mouse strain. Specifically, we showed that the depletion of DCs led to increased viral spread into the nervous system, resulting in an increased rate of morbidity and mortality. Furthermore, we showed that ablation of DCs impaired the optimal activation of NK cells and CD4+ and CD8+ T cells in response to HSV-1. These data demonstrated that DCs were essential not only in the optimal activation of the acquired T-cell response to HSV-1 but also that DCs were crucial for innate resistance to HSV-1 infection.

Gene Therapy in a Humanized Mouse Model of Familial Hypercholesterolemia Leads to Marked Regression of Atherosclerosis

Background Familial hypercholesterolemia (FH) is an autosomal codominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Homozygous FH patients (hoFH) have severe hypercholesterolemia leading to life threatening atherosclerosis in childhood and adolescence. Mice with germ line interruptions in the Ldlr and Apobec1 genes (Ldlr−/−Apobec1−/−) simulate metabolic and clinical aspects of hoFH, including atherogenesis on a chow diet. Methods/Principal Findings In this study, vectors based on adeno-associated virus 8 (AAV8) were used to deliver the gene for mouse Ldlr (mLDLR) to the livers of Ldlr−/−Apobec1−/− mice. A single intravenous injection of AAV8.mLDLR was found to significantly reduce plasma cholesterol and non-HDL cholesterol levels in chow-fed animals at doses as low as 3×109 genome copies/mouse. Whereas Ldlr−/−Apobec1−/− mice fed a western-type diet and injected with a control AAV8.null vector experienced a further 65% progression in atherosclerosis over 2 months compared with baseline mice, Ldlr−/−Apobec1−/− mice treated with AAV8.mLDLR realized an 87% regression of atherosclerotic lesions after 3 months compared to baseline mice. Immunohistochemical analyses revealed a substantial remodeling of atherosclerotic lesions. Conclusions/Significance Collectively, the results presented herein suggest that AAV8-based gene therapy for FH may be feasible and support further development of this approach. The pre-clinical data from these studies will enable for the effective translation of gene therapy into the clinic for treatment of FH.

Characterization of age-related changes in natural killer cells during primary influenza infection in mice.

The current investigation examined the importance of natural killer (NK) cells during the innate immune response to primary influenza infection in young and aged mice. Young (6-8 weeks) and aged (22 months) C57BL/6 mice were infected intranasally with influenza A virus, and NK cell-mediated cytotoxicity was determined in lung and spleen during the first 4 days of infection. Aged mice demonstrated both a decrease in influenza-inducible NK activity and a reduction in the percentage and number of NK1.1+ cells in response to primary influenza infection, relative to young mice. In order to further establish a role for NK cells in controlling influenza infection, young mice were depleted of NK cells in vivo by injecting rabbit anit-NK1.1 antibody 2 days and 1 day prior to influenza infection. Young mice depleted of NK cells exhibited increased weight loss and lung virus titers during the course of infection, compared to young mice infected with influenza virus. These data indicate that NK cell function is impaired in response to primary influenza infection in aged mice. More importantly, these results underscore the essential role of NK cells in controlling virus titers in lung during the early course of influenza infection, regardless of age.

CD4+ T Cells Are Required for the Priming of CD8+ T Cells following Infection with Herpes Simplex Virus Type 1

ABSTRACT The role of CD4+ helper T cells in modulating the acquired immune response to herpes simplex virus type 1 (HSV-1) remains ill defined; in particular, it is unclear whether CD4+ T cells are needed for the generation of the protective HSV-1-specific CD8+-T-cell response. This study examined the contribution of CD4+ T cells in the generation of the primary CD8+-T-cell responses following acute infection with HSV-1. The results demonstrate that the CD8+-T-cell response generated in the draining lymph nodes of CD4+-T-cell-depleted C57BL/6 mice and B6-MHC-II−/− mice is quantitatively and qualitatively distinct from the CD8+ T cells generated in normal C57BL/6 mice. Phenotypic analyses show that virus-specific CD8+ T cells express comparable levels of the activation marker CD44 in mice lacking CD4+ T cells and normal mice. In contrast, CD8+ T cells generated in the absence of CD4+ T cells express the interleukin 2 receptor α-chain (CD25) at lower levels. Importantly, the CD8+ T cells in the CD4+-T-cell-deficient environment are functionally active with respect to the expression of cytolytic activity in vivo but exhibit a diminished capacity to produce gamma interferon and tumor necrosis factor alpha. Furthermore, the primary expansion of HSV-1-specific CD8+ T cells is diminished in the absence of CD4+-T-cell help. These results suggest that CD4+-T-cell help is essential for the generation of fully functional CD8+ T cells during the primary response to HSV-1 infection.

Dendritic Cells Are Required for Optimal Activation of Natural Killer Functions following Primary Infection with Herpes Simplex Virus Type 1

ABSTRACT Natural killer (NK) cells play an important role in the optimal clearance of herpes simplex virus type 1 (HSV-1) infection in mice. Activated NK cells function via cytokine secretion or direct cytolysis of target cells; dendritic cells (DCs) are thought to make critical contributions in the activation of both of these functions. Yet, the magnitude and physiological relevance of DC-mediated NK cell activation in vivo is not completely understood. To examine the contribution of DC help in regulating NK cell functions after infection with HSV-1, we utilized a transgenic mouse model that allows the transient ablation of DCs. Using this approach, it was found that the gamma interferon (IFN-γ) expression potential of NK cells is quantitatively and qualitatively impaired in the absence of DCs. With regard to priming of NK cytolytic functions, the ablation of DCs did not significantly affect cytotoxic protein expression by NK cells. An in vivo cytolytic assay did, however, reveal impairments in the magnitude of NK cell cytotoxicity. Overall, this study provides direct evidence that functional DCs are required for optimal IFN-γ expression and cytolytic function by NK cells following infection with HSV-1.

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.

Gene therapy for dyslipidemia: a review of gene replacement and gene inhibition strategies

Abstract Despite numerous technological and pharmacological advances and more detailed knowledge of molecular etiologies, cardiovascular diseases remain the leading cause of morbidity and mortality worldwide claiming over 17 million lives a year. Abnormalities in the synthesis, processing and catabolism of lipoprotein particles can result in severe hypercholesterolemia, hypertriglyceridemia or low HDL‑C. Although a plethora of antidyslipidemic pharmacological agents are available, these drugs are relatively ineffective in many patients with Mendelian lipid disorders, indicating the need for new and more effective interventions. In vivo somatic gene therapy is one such intervention. This article summarizes current strategies being pursued for the development of clinical gene therapy for dyslipidemias that cannot effectively be treated with existing drugs.

From lipoproteins to chondrocytes: a brief summary of the European Medicines Agency's regulatory guidelines for advanced therapy medicinal products.

Since June 2012, the European Union (EU) has witnessed two major regulatory milestones in the fields of gene, cell, and tissue therapy. In July 2012, the European Medicines Agency (EMA) approved the Western Worlds first-ever gene therapy product (Glybera [alipogene tiparvovec]; uniQure, Amsterdam, The Netherlands); more recently, in April 2013, the EMA approved the EUs first-ever combined tissue-engineered product (MACI [matrix-induced autologous chondrocyte implantation]; Genzyme, Cambridge, MA). The recent spate of positive clinical results in both gene and cell therapy suggests that there will be more of these approvals in the near future (European Medicines Agency [A]). This brief commentary uses Glybera, MACI, and the first-ever EMA-approved cell therapy product (ChondroCelect; TiGenix, Leuven, Belgium) as case studies to elucidate the EMAs current regulatory guidelines for the development of gene, cell, and tissue therapy products. Based in London, the EMA was set up in 1995 with funding from the EU and the pharmaceutical industry. The Committee for Medicinal Products for Human Use (CHMP) was established by the EMA in 2004 and is responsible for preparing official opinions on all questions concerning medicines for human use. If the CHMP concludes that the quality, safety, and efficacy of the medicinal product are sufficiently proven, it adopts a positive opinion. The positive opinion is sent to the European Commission, where it is transformed into a marketing authorization (MA) valid in Iceland, Liechtenstein, Norway, and all EU member countries. This allows the MA holder to market the medicine and make it available to patients and health care professionals throughout Europe. Until 2007, the term “medicines” broadly applied to all products of chemical (i.e., small molecules) and biological (i.e., monoclonal antibodies) origin. In 2008, the EMA developed a consolidated regulatory framework specific for advanced therapy medicinal products (ATMPs). ATMPs comprise gene therapy, somatic cell therapy, and tissue-engineered products. To assist in the implementation of ATMP specific guidelines, the EMA established the Committee for Advanced Therapies (CAT) in 2009 (European Medicines Agency [B]). The CAT is an independent multidisciplinary committee that functions in various capacities along the regulatory pathway. Before submitting a marketing authorization application to the EMA, an ATMP sponsor may engage with the CAT at any step in the clinical development pathway (Fig. 1). These discussions are not legally required but provide ATMP sponsors a valuable opportunity to obtain guidance from the CAT on a myriad of issues including ATMP classifications, scientific advice requests, and collaboration with the EMAs Innovation Task Force. FIG. 1. Committee for Advanced Therapies (CAT) specific involvement in regulation of advanced therapy medicinal products (ATMPs) before submission of a marketing authorization application (MAA). Adapted from European Medicines Agency ([A] and [C]). After submission of a marketing authorization application (MAA) (Fig. 2), the CATs primary responsibilities are to assess the quality, safety, and efficacy of ATMPs and to provide the CHMP with a recommendation for approval or refusal of the MAA. EMA regulations mandate that the CHMP reach a formal decision on approval/refusal within 210 days of submission of the MAA; the clock is stopped, if necessary, to ask the applicant for clarification or further supporting data. The CAT publishes a monthly report on all of its activities. According to the April 2013 report a total of 10 MAAs have been submitted since 2009 (European Medicines Agency [C]). Of these applications, there have been four positive draft opinions, which have resulted in marketing authorizations for three ATMP products (ChondroCelect, Glybera, and MACI), two negative draft opinions, and four withdrawals. There are currently three pending MAAs. FIG. 2. Timeline outlining Committee for Advanced Therapies (CAT) and Committee for Medicinal Products for Human Use (CHMP) specific involvement in regulation of advanced therapy medicinal products (ATMPs) after submission of marketing authorization application ... The first gene therapy product to receive a positive opinion from the CAT and the CHMP was Glybera, an adeno-associated viral vector encoding the gene for lipoprotein lipase (LPL) protein (European Medicines Agency [D]). LPL deficiency is a rare (i.e., orphan disease) autosomal recessive inherited condition caused by homozygosity or compound heterozygosity for mutations in the LPL gene; LPL deficiency affects approximately one or two people per million. In March 2004, Glybera was granted an orphan medicinal product designation. This designation allows a pharmaceutical company, in this case Amsterdam Molecular Therapeutic (AMT, Amsterdam, The Netherlands), to benefit from incentives from the European Union to develop a medicine for a rare disease, such as reduced fees and protection from competition once on the market. The EMAs Committee for Orphan Medicinal Products (COMP) examines applications for orphan designations. AMT subsequently submitted an MAA for Glybera in December 2009. In June 2011, the CHMP and the CAT adopted negative opinions on the MAA, concluding that AMT had not provided sufficient evidence of a persistence of effect in lowering blood fats in a clinically relevant manner and that there were too few patients for whom sufficiently long-term data were available. During reexamination of the MAA, at AMTs request, the CAT considered that there was scope for approval of Glybera with additional postmarketing studies, but the CHMP maintained its negative opinion in October 2011. In January 2012, during a meeting of the Member States Standing Committee on human medicinal products, the European Commission (EC) asked the EMA to reevaluate the application for Glybera in a restricted group of patients with severe or multiple pancreatitis attacks due to LPL deficiency. After detailed scientific discussions in both committees, the CAT adopted a positive draft opinion in June 2012, which was then endorsed by the CHMP in July 2012. The CHMPs positive opinion on Glybera was subsequently sent to the EC for adoption of the MA. AMT was acquired by uniQure in April 2012 and currently holds the MA for Glybera. The MA allows Glybera to be intramuscularly injected, at 1×1012 genome copies/kg body weight, in patients diagnosed with LPL deficiency and suffering from severe or multiple pancreatitis attacks. It should be noted that Glybera was granted an MA under exceptional circumstances, meaning that it will need to meet certain specific obligations and will have to be reviewed annually by the EMA (European Medicines Agency [D]). Two ATMPs have been approved by EMA for the treatment of full-thickness articular cartilage defects of the knee. ChondroCelect (TiGenix) was the first-ever cellular product to complete its developmental cycle, from research to clinical approval, as an ATMP under the guidance of the EMA (European Medicines Agency [E]). ChondroCelect is manufactured from autologous chondrocytes isolated from biopsy material that is subsequently positioned in the cartilage defect with a collagen membrane. TiGenix submitted their application for licensing in June 2007. In June 2009, the CAT offered a positive draft opinion based on the data submitted and discussion within the scientific committee, which was subsequently endorsed by CHMP, resulting in the granting of market authorization. A second chondrocyte-derived ATMP, MACI (Genzyme), recently became the first combined tissue-engineered product to receive CHMP approval after a positive recommendation by the CAT (European Medicines Agency [F]). MACI is also produced from isolated autologous chondrocytes, but differs from ChondroCelect in that the autologous chondrocytes are seeded on a three-dimensional cell-free porcine scaffold for implantation. The seeded scaffold is then shipped to the surgical center, where it is trimmed to match the articular cartilage defect, and cemented in position with a fibrin glue. MACI was found to be superior to microfracture in improving pain and joint function in an open randomized trial. The benefit-to-risk ratio was considered positive and therefore the CHMP granted marketing authorization for MACI in April 2013. Before consideration by the CAT, MACI had been available since 1998 in a number of European countries including Austria, Belgium, Denmark, Germany, Greece, Ireland, Italy, Holland, Norway, Portugal, Spain, and the United Kingdom, in accordance with local regulations. This year, the CAT expects to review three or four MAAs for ATMPs. This compares with three initial applications for MAA received in 2012, which led to the authorization of Glybera. Although the number of MAAs for advanced therapies is still limited, the research and development pipeline is large (European Medicines Agency [A]). As a consequence, a high number of MAAs for advanced therapies is expected over the next 5 to 10 years (Maciulaitis et al., 2012). Among the ATMPs under development, three-quarters are cell-based medicinal products with the remainder representing gene therapies. Most of these products are being developed for cancers, cardiovascular diseases, and hematology-related conditions. Interestingly, according to the EMA, most of these ATMPs are under development by noncommercial sponsors (60%) and micro-, small, and medium-sized enterprises (SMEs; 38%) (European Medicines Agency [A]; Maciulaitis et al., 2012).

Identification and functional characterization in vivo of a novel splice variant of LDLR in rhesus macaques

In the course of developing a low-density lipoprotein receptor (LDLR) gene therapy treatment for homozygous familial hypercholesterolemia (HoFH), we planned to examine the efficacy in a nonhuman primate model, the rhesus macaque heterozygous for an LDL receptor mutation fed a high-fat diet. Unexpectedly, our initial cDNA sequencing studies led to the identification of a heretofore unidentified splicing isoform of the rhesus LDLR gene. Compared with the publicly available GenBank reference sequence of rhesus LDLR, the novel isoform contains a 21 bp in frame insertion. This sequence coincides with part of exon 5 and creates a site for the restriction enzyme MscI. Using this site as a marker for the 21 bp in-frame insertion, we conducted a restriction enzyme screen to examine for the prevalence of the novel isoform in rhesus liver tissue cDNA and its homolog in human liver tissue cDNA. We found that the novel isoform is the predominant LDLR cDNA found in rhesus liver and the sole LDLR cDNA found in human liver. Finally, we compared the in vivo functionality of the novel and previously identified rhesus LDLR splicing isoforms in a mouse model of HoFH.