Babak Moghimi

Oral delivery of bioencapsulated coagulation factor IX prevents inhibitor formation and fatal anaphylaxis in hemophilia B mice

To address complications of pathogenic antibody or life-threatening anaphylactic reactions in protein replacement therapy for patients with hemophilia or other inherited protein deficiencies, we have developed a prophylactic protocol using a murine hemophilia B model. Oral delivery of coagulation factor IX fused with cholera toxin β-subunit (with or without a furin cleavage site; CTB-FFIX or CTB-FIX), expressed in chloroplasts (up to 3.8% soluble protein or 0.4 mg/g leaf tissue), bioencapsulated in plant cells, effectively blocked formation of inhibitory antibodies (undetectable or up to 100-fold less than controls). Moreover, this treatment eliminated fatal anaphylactic reactions that occurred after four to six exposures to intravenous F.IX. Whereas only 20–25% of control animals survived after six to eight F.IX doses, 90–93% of F.IX-fed mice survived 12 injections without signs of allergy or anaphylaxis. Immunostaining confirmed delivery of F.IX to Peyers patches in the ileum. Within 2–5 h, feeding of CTB-FFIX additionally resulted in systemic delivery of F.IX antigen. This high-responder strain of hemophilia B mice represents a new animal model to study anaphylactic reactions. The protocol was effective over a range of oral antigen doses (equivalent to 5–80 μg recombinant F.IX/kg), and controlled inhibitor formation and anaphylaxis long-term, up to 7 months (∼40% life span of this mouse strain). Oral antigen administration caused a deviant immune response that suppressed formation of IgE and inhibitory antibodies. This cost-effective and efficient approach of antigen delivery to the gut should be applicable to several genetic diseases that are prone to pathogenic antibody responses during treatment.

The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9-dependent innate immune responses in the liver.

Although adeno-associated viral (AAV) vectors have been successfully used in hepatic gene transfer for treatment of hemophilia and other diseases in animals, adaptive immune responses blocked long-term transgene expression in patients on administration of single-stranded AAV serotype-2 vector. More efficient vectors have been developed using alternate capsids and self-complimentary (sc) genomes. This study investigated their effects on the innate immune profile on hepatic gene transfer to mice. A mild and transient up-regulation of myeloid differentiation primary response gene (88), TLR9, TNF-α, monocyte chemotactic protein-1, IFN-γ inducible protein-10, and IFN-α/β expression in the liver was found after single-stranded AAV vector administration, regardless of the capsid sequence. In contrast, scAAV vectors induced higher increases of these transcripts, upregulated additional proinflammatory genes, and increased circulating IL-6. Neutrophil, macrophage, and natural killer cell liver infiltrates were substantially higher on injection of scAAV. Some but not all of these responses were Kupffer cell dependent. Independent of the capsid or expression cassette, scAAV vectors induced dose-dependent innate responses by signaling through TLR9. Increased innate responses to scAAV correlated with stronger adaptive immune responses against capsid (but not against the transgene product). However, these could be blunted by transient inhibition of TLR9.

Impact of the underlying mutation and the route of vector administration on immune responses to factor IX in gene therapy for hemophilia B.

Immune responses to factor IX (F.IX), a major concern in gene therapy for hemophilia, were analyzed for adeno-associated viral (AAV-2) gene transfer to skeletal muscle and liver as a function of the F9 underlying mutation. Vectors identical to those recently used in clinical trials were administered to four lines of hemophilia B mice on a defined genetic background [C3H/HeJ with deletion of endogenous F9 and transgenic for a range of nonfunctional human F.IX (hF.IX) variants]. The strength of the immune response to AAV-encoded F.IX inversely correlated with the degree of conservation of endogenous coding information and levels of endogenous antigen. Null mutation animals developed T- and B-cell responses in both protocols. However, inhibitor titers were considerably higher upon muscle gene transfer (or protein therapy). Transduced muscles of Null mice had strong infiltrates with CD8+ cells, which were much more limited in the liver and not seen for the other mutations. Sustained expression was achieved with liver transduction in mice with crm(-) nonsense and missense mutations, although they still formed antibodies upon muscle gene transfer. Therefore, endogenous expression prevented T-cell responses more effectively than antibody formation, and immune responses varied substantially depending on the protocol and the underlying mutation.

Induction of tolerance to factor VIII by transient co-administration with rapamycin.

See also Miao CH. Tilt balance towards regulation: evolving new strategy for treatment of hemophilia inhibitors. This issue, pp 1521–3.DOI:10.1111/j.1538‐7836.2011.04351.x.

Tolerance Induction to Factor VIII by Transient Co-administration with Rapamycin

See also Miao CH. Tilt balance towards regulation: evolving new strategy for treatment of hemophilia inhibitors. This issue, pp 1521–3.DOI:10.1111/j.1538‐7836.2011.04351.x.

Androgen-induced cerebral venous sinus thrombosis in a young body builder: case report

BackgroundCerebral venous sinus thrombosis is an infrequent disease with a variety of causes. Pregnancy, puerperium, contraceptive pills and intracranial infections are the most common causes. The patient may present with headache, focal neurological deficits and seizures.The clinical outcome is highly variable and treatment with heparin is advised.Case presentationThe patient is a 22 year old male who presented with headache, repeated vomiting and papilledema.He was a bodybuilder doing exercise since 5 years ago, who had used nandrolone decaonoate 25 milligrams intramuscularly during the previous 5 months. Brain MRI and MRV showed superior sagital and transverse sinus thrombosis and extensive investigations did not reveal any known cause.ConclusionsWe suggested that androgen was the predisposing factor in our patient. Androgens may increase coagulation factors or platelet activity and cause arterial or venous thrombosis.As athletes may hide using androgens it should be considered as a predisposing factor for thrombotic events in such patients.

An Iranian family with Alzheimer’s disease caused by a novel APP mutation (Thr714Ala)

Despite the fact that APP mutations were the first described cause of AD,1 relatively few pathogenic mutations have been described. In this article, we describe the occurrence of a novel APP mutation (T714A) in an Iranian family with nine documented affected individuals in three generations. The phenotype in this family is similar to that of many of the other families with APP mutations, with an average onset age of approximately 55 years. A family with multiple members affected by progressive dementia was identified in northern Iran (see the figure). The average age at onset of dementia was in approximately 55 years with a disease progression lasting 2 to 10 years. The clinical features and age at onset were reminiscent of the first family described with APP mutations.1,2⇓ We briefly describe the clinical features of the family and the identification of the pathogenic mutation and discuss the …

Locus control region mediated regulation of adult β-globin gene expression

Many genes residing in gene clusters and expressed in a differentiation or developmental‐stage specific manner are regulated by locus control regions (LCRs). These complex genetic regulatory elements are often composed of several DNAse I hypersensitive sites (HS sites) that function together to regulate the expression of several cis‐linked genes. Particularly well characterized is the LCR associated with the β‐globin gene locus. The β‐globin LCR consists of five HS sites that are located upstream of the β‐like globin genes. Recent data demonstrate that the LCR is required for the association of the β‐globin gene locus with transcription foci or factories. The observation that RNA polymerase II associates with the LCR in erythroid progenitor or hematopoietic stem cells which do not express the globin genes suggests that the LCR is always in an accessible chromatin configuration during differentiation of erythroid cells. We propose that erythroid specific factors together with ubiquitous proteins mediate a change in chromatin configuration that juxtaposes the globin genes and the LCR. The proximity then facilitates the transfer of activities from the LCR to the globin genes. In this article we will discuss recent observations regarding β‐globin locus activation with a particular emphasis on LCR mediated activation of adult β‐globin gene expression. J. Cell. Biochem. 105: 9–16, 2008.

Prevention and Reversal of Antibody Responses Against Factor IX in Gene Therapy for Hemophilia B

Intramuscular (IM) administration of an adeno-associated viral (AAV) vector represents a simple and safe method of gene transfer for treatment of the X-linked bleeding disorder hemophilia B (factor IX, F.IX, deficiency). However, the approach is hampered by an increased risk of immune responses against F.IX. Previously, we demonstrated that the drug cocktail of immune suppressants rapamycin, IL-10, and a specific peptide (encoding a dominant CD4+ T cell epitope) caused an induction of regulatory T cells (Treg) with a concomitant apoptosis of antigen-specific effector T cells (Nayak et al., 2009). This protocol was effective in preventing inhibitory antibody formation against human F.IX (hF.IX) in muscle gene transfer to C3H/HeJ hemophilia B mice (with targeted F9 gene deletion). Here, we show that this protocol can also be used to reverse inhibitor formation. IM injection of AAV1–hF.IX vector resulted in inhibitors of on average 8–10 BU within 1 month. Subsequent treatment with the tolerogenic cocktail accomplished a rapid reduction of hF.IX-specific antibodies to <2 BU, which lasted for >4.5 months. Systemic hF.IX expression increased from undetectable to >200 ng/ml, and coagulation times improved. In addition, we developed an alternative prophylactic protocol against inhibitor formation that did not require knowledge of T cell epitopes, consisting of daily oral administration of rapamycin for 1-month combined with frequent, low-dose intravenous injection of hF.IX protein. Experiments in T cell receptor transgenic mice showed that the route and dosing schedule of drug administration substantially affected Treg induction. When combined with intravenous antigen administration, oral delivery of rapamycin had to be performed daily in order to induce Treg, which were suppressive and phenotypically comparable to natural Treg.

Recruitment of coregulator complexes to the β‐globin gene locus by TFII‐I and upstream stimulatory factor

Upstream stimulatory factor and TFII‐I are ubiquitously expressed helix‐loop‐helix transcription factors that interact with E‐box sequences and or initiator elements. We previously demonstrated that upstream stimulatory factor is an activator of β‐globin gene expression whereas TFII‐I is a repressor. In the present study, we demonstrate that upstream stimulatory factor interacts with the coactivator p300 and that this interaction is restricted to erythroid cells expressing the adult β‐globin gene. Furthermore, we demonstrate that Suz12, a component of the polycomb repressor complex 2, is recruited to the β‐globin gene. Reducing expression of Suz12 significantly activates β‐globin gene expression in an erythroid cell line with an embryonic phenotype. Suz12 also interacts with the adult β‐globin gene during early stages of erythroid differentiation of mouse embryonic stem cells. Our data suggest that TFII‐I contributes to the recruitment of the polycomb repressor complex 2 complex to the β‐globin gene. Together, these data demonstrate that the antagonistic activities of upstream stimulatory factor and TFII‐I on β‐globin gene expression are mediated at least in part by protein complexes that render the promoter associated chromatin accessible or inaccessible for the transcription complex.