Strategy for treating motor neuron diseases using a fusion protein of botulinum toxin binding domain and streptavidin for viral vector access: work in progress.

Although advances in understanding of the pathogenesis of amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) have suggested attractive treatment strategies, delivery of agents to motor neurons embedded within the spinal cord is problematic. We have designed a strategy based on the specificity of botulinum toxin, to direct entry of viral vectors carrying candidate therapeutic genes into motor neurons. We have engineered and expressed fusion proteins consisting of the binding domain of botulinum toxin type A fused to streptavidin (SAv). This fusion protein will direct biotinylated viral vectors carrying therapeutic genes into motor nerve terminals where they can enter the acidified endosomal compartments, be released and undergo retrograde transport, to deliver the genes to motor neurons. Both ends of the fusion proteins are shown to be functionally intact. The binding domain end binds to mammalian nerve terminals at neuromuscular junctions, ganglioside GT1b (a target of botulinum toxin), and a variety of neuronal cells including primary chick embryo motor neurons, N2A neuroblastoma cells, NG108-15 cells, but not to NG CR72 cells, which lack complex gangliosides. The streptavidin end binds to biotin, and to a biotinylated Alexa 488 fluorescent tag. Further studies are in progress to evaluate the delivery of genes to motor neurons in vivo, by the use of biotinylated viral vectors.

Rebooting the immune system with high-dose cyclophosphamide for treatment of refractory myasthenia gravis.

A small but important proportion of patients with myasthenia gravis (MG) are refractory to conventional immunotherapy. We have treated 12 such patients by "rebooting" the immune system with high-dose cyclophosphamide (Hi Cy, 200 mg/kg), which largely eliminates the mature immune system, while leaving hematopoietic precursors intact. The objective of this report is to describe the clinical and immunologic results of Hi Cy treatment of refractory MG. We have followed 12 patients clinically for 1-9 years, and have analyzed their humoral and cellular immunologic parameters. Hi Cy is safe and effective. All but one of the patients experienced dramatic clinical improvement for variable periods from 5 months to 7.5 years, lasting for more than 1 year in seven of the patients. Two patients are still in treatment-free remission at 5.5 and 7.5 years, and five have achieved responsiveness to immunosuppressive agents that were previously ineffective. Hi Cy typically reduced, but did not completely eliminate, antibodies to the autoantigen AChR or to tetanus or diphtheria toxin; re-immunization with tetanus or diphtheria toxoid increased the antibody levels. Despite prior thymectomy, T cell receptor excision circles, generally considered to reflect thymic emigrant T cells, were produced by all patients. Hi Cy treatment results in effective, but often not permanent, remission in most refractory myasthenic patients, suggesting that the immune system is in fact "rebooted," but not "reformatted." We therefore recommend that treatment of refractory MG with Hi Cy be followed with maintenance immunotherapy.

Clinical comparison of muscle-specific tyrosine kinase (MuSK) antibody-positive and -negative myasthenic patients.

We assayed cryopreserved sera from 38 acetylcholine receptor (AChR) antibody-negative patients with myasthenia gravis (MG) who were followed clinically for muscle-specific tyrosine kinase (MuSK) antibodies and analyzed and compared their clinical characteristics. None of 13 sera from patients with purely ocular MG were positive. Sera from 10 of 25 patients (40%) with generalized MG were positive for MuSK antibodies. The age at onset of myasthenic symptoms was significantly earlier in MuSK antibody-positive patients (P = 0.02). MuSK antibodies were present in AChR antibody-negative patients of either gender, with virtually identical prevalence in women (41.2%) and men (37.5%). The distribution of weakness more commonly involved neck muscles in MuSK antibody-positive patients, and limb muscles in MuSK antibody-negative patients. Patients responded to immunosuppressive treatment regardless of whether MuSK antibody was present. We conclude that MuSK antibodies are present and diagnostically useful in a subset of myasthenic patients without AChR antibodies. Although the distribution of weakness differs somewhat depending on whether MuSK antibodies are present, responses to anticholinesterase and immunosuppressive treatments are similar.

T cell receptor transgenic mice recognizing the immunodominant epitope of the Torpedo californica acetylcholine receptor.

Myasthenia gravis (MG) is an autoimmune disease caused by T cell-dependent antibody-mediated reduction of acetylcholine receptors (AChR) at the neuromuscular junction. Immunization of animals with Torpedo californica AChR (TAChR) results in an experimental model of MG. We used the variable regions of alpha and beta T cell receptor (TCR) genes recognizing an immunodominant peptide containing amino acids 146-162 from the alpha subunit of TAChR presented in the context of I-A(b) to generate TCR-transgenic mice. We found that the transgenic TCR was strongly positively selected and that transgenic T cells proliferated robustly to the immunodominant peptide and TAChR. Unexpectedly, there was a variable paucity of B cells in the blood and spleen from transgenic mice, which averaged about 16% of peripheral blood lymphocytes, compared to 55% in wild-type B6 mice. Unselected transgenic mice immunized with TAChR exhibited weak anti-TAChR antibody responses. However, transgenic mice selected to have relatively higher B cell numbers produced anti-TAChR titers equal to B6 mice and a predominance of Th1-induced antibody isotypes were observed in certain experiments. The incidence and severity of clinical disease was variable following immunizations. These mice should be useful for studying the pathogenesis and treatment of MG.