A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy

Background  

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation.     

Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal: lack of genotype/phenotype correlations

It has previously been shown that, in the heterozygous state, mutations in the SOX9 gene cause campomelic dysplasia (CD) and the often associated autosomal XY sex reversal. In 12 CD patients, 10 novel mutations and one recurrent mutation were characterized in one SOX9 allele each, and in one case, no mutation was found. Four missense mutations are all located within the high mobility group (HMG) domain. They either reduce or abolish the DNA-binding ability of the mutant SOX9 proteins. Among the five nonsense and three frameshift mutations identified, two leave the C-terminal transactivation (TA) domain encompassing residues 402-509 of SOX9 partly or almost completely intact. When tested in cell transfection experiments, the recurrent nonsense mutation Y440X, found in two patients who survived for four and more than 9 years, respectively, exhibits some residual transactivation ability. In contrast, a frameshift mutation extending the protein by 70 residues at codon 507, found in a patient who died shortly after birth, showed no transactivation. This is apparently due to instability of the mutant SOX9 protein as demonstrated by Western blotting. Amino acid substitutions and nonsense mutations are found in patients with and without XY sex reversal, indicating that sex reversal in CD is subject to variable penetrance. Finally, none of 18 female patients with XY gonadal dysgenesis (Swyer syndrome) showed an altered SOX9 banding pattern in SSCP assays, providing evidence that SOX9 mutations do not usually result in XY sex reversal without skeletal malformations.      

Cloning of murine IL-22 receptor alpha 2 and comparison with its human counterpart

We have identified the mouse and rat homologs of human interleukin-22 receptor alpha 2 (IL-22R alpha 2) and compared the localization, structure, and expression of the encoding murine and human genes. The mouse IL-22R alpha 2-encoding gene is located on chromosome 10A3 between, like in human, the genes for interferon-gamma R1 and IL-20R1. It spans a region of approximately 10 kb therefore being three times shorter than the human gene. Although the overall gene structure in both species is similar, the mouse gene lacks a counterpart to the third coding exon of the human gene known to be alternatively spliced. Like in human, mouse and rat IL-22R alpha 2 exist only as soluble receptors as deduced from the lack of transmembrane and intracellular domains encoding sequences. Quantitative expression analyses showed, analogically to the human system, a limited tissue distribution of mouse IL-22R alpha 2 mRNA. Differential modulation of IL-22R alpha 2 mRNA expression was observed upon systemic inflammation in mice in spleen, thymus, and lymph node.     

Improvements in Gene Therapy

Gene therapy is an interesting approach for the correction of defective genes, the treatment of cancer and the introduction of immunomodulatory genes. Various techniques for gene transfer into cells or tissues have been developed within the last decade; these can be divided generally into viral and nonviral gene transfer systems. Nonviral techniques include the liposome- or gene gun-mediated introduction of therapeutic genes; however, the efficiency of gene transfer by these applications is still very low. In contrast, viruses have optimised their strategies for efficient infection of virtually any cell type in a mammalian organism. The genetic modification of genomes from different virus families (Adenoviridae, Retroviridae, Herpesviridae) led to the development of gene therapy vectors with a similar capacity to infect cells or tissues as that of wild type viruses. In contrast to wild type viruses, gene therapy vectors are engineered to transfer therapeutic genes into the target cells or tissues. In addition, they have lost their capacity for replication in target cells, because of the removal of essential genes, which allows replication only in specialised packaging cell lines engineered for the production of recombinant viruses. Despite considerable progress over the past decade in the generation of gene transfer systems with reduced immunogenic properties, the remaining immunogenicity of many gene therapy vectors is still the major hurdle, preventing their frequent application in clinical trials. Recombinant adenoviruses have been shown to be promising vectors for gene therapy, since they are able to transduce both quiescent and proliferating cells very efficiently. However, a major disadvantage of adenoviral vectors lies in the activation of both the innate and adaptive parts of the recipient's immune system when applied in vivo. The inflammatory responses induced by adenovirus particles can be very strong and can be fatal in patients treated with these adenoviral constructs. Therefore, many experiments have been performed in the effort to prevent these inflammatory responses mediated by adenoviral particles. The depletion of cell populations responsible for these inflammatory responses as well as the application of immunosuppressive drugs have been investigated. Moreover, the generation of less immunogenic adenoviral vectors by further genetic modification within the adenoviral genome has led to vectors with reduced immunogenic properties. Both strategies to reduce inflammatory responses against adenoviral particles are discussed in this review.     

Damage of purkinje cell axons following chronic phenytoin administration: An animal model of distal axonopathy

Summary An animal model of central distal axonopathy following chronic administration of phenytoin is described. Male C57/BL6J mice received diphenylhydantoin (DPH) in the daily diet (liquid diet Stardit, supplemented with vitamins) over a period of 8 weeks. Control and experimental animals were pair-fed.Twelve mice of both groups were perfused via the left ventricle with glutaraldehyde. Representative samples of the cerebral cortex (area 3), cerebellum (vermis and deep cerebellar nuclei), thalamus, hypothalamus, and liver were embedded in araldite. Semithin sections and electron microscopy of the cerebellar vermis revealed marked dystrophic changes in the Purkinje cell axons. The presynaptic segments of Purkinje cell axons in the deep cerebellar nuclei showed massive enlargement and swelling due to accumulation of spherical particles and tubular structures in the axoplasm. These structures represent a proliferation of the smooth endoplasmic reticulum.Identical changes were found in hepatocytes of treated animals. Because phenytoin induces hepatic microsomal enzymes, we suggest that phenytoin-related Purkinje cell damage may be produced by an induction of Purkinje cell microsomes with proliferation of the smooth endoplasmic reticulum which causes a swelling and enlargement of presynaptic segments of Purkinje cell axons in deep cerebellar nuclei. Chronic phenytoin administration to mice is a new model of phenytoin-induced encephalopathy and of distal axonopathy of cerebellar neurons.Supported by the Deutsche ForschungsgemeinschaftPresented in Part at the Joint Meeting of the German and Scandinavian Neuropathologists, Turku, Finland, June 3–4, 1983     

Dynamic and static phantoms for evaluation of digital subtraction angiographic systems

Two types of phantoms were developed with which to evaluate the overall performance of digital subtraction angiography (DSA) systems. A dynamic phantom, called a "fish bone" phantom, consists of polyethylene tubes that simulate blood vessels with various lesions, such as stenoses, ulcers, and aneurysms. With this phantom, washout curves were obtained representing the relationship between iodine content and time. It will be useful for qualitative assessment of DSA images, evaluation of different image-processing schemes, and studies of blood flow analysis. A static phantom, called a "C-D" phantom, can be used for measurement of quantitative contrast-detail (C-D) diagrams and for daily monitoring of DSA systems. This was constructed of tubes of seven different diameters (2.15-0.28 mm) and 14 different concentrations of contrast medium (100%-1.1% Renografin-76 [meglumine and sodium diatrizoate]). The C-D diagrams were determined from an observer performance study using C-D phantom images obtained at four different DSA settings.     

CT stereotactic biopsy in the differential diagnosis of deeply situated intracerebral hematomas

The diagnosis of intracerebral haematomas, especially of those which are relatively small, occupy little space and are deeply situated, presents considerable problems. The problem is even greater when the expected acute case history and the acute beginning of the symptoms do not occur and unusual localisations are found. The consequences of this are false diagnoses and the treatment of these patients within the framework of blanket diagnosis "intracerebral tumours" or "space occupying processes" without any confirmation of the histological diagnosis. - Using a sample of 26 patients where the histological diagnosis of non-recent intracerebral hemorrhages had been confirmed (out of a series of 818 CT-stereotactically biopsied patients punctured by us from the beginning of 1983 until the end of 1984), the problem of establishing a diagnosis is exposed. - A histological diagnoses should in any case be confirmed before any thorough and deep-reaching therapy is begun, since false diagnoses and misinterpretations can cause serious consequences for the patient.     

Needle biopsy of renal allografts: comparison of two techniques

Two techniques for renal allograft biopsy were retrospectively evaluated to compare relative safety and efficacy. After ultrasound (US) localization of the kidney and biopsy with a hand-held 14-gauge cutting needle, an adequate specimen was obtained in 74 of 77 cases (96%). Major complications occurred in six of these 77 cases (8%). One hundred four biopsies were performed by using a smaller 18-gauge cutting needle with a spring-loaded biopsy "gun" and real-time US guidance. With this newer technique, specimens adequate for diagnosis were obtained in 99 biopsies (95%). There was a single major complication with this technique (1%). The 18-gauge needle with real-time US guidance yields comparably adequate specimens with a lower frequency of complications.