Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.

The essential product of the Duchenne muscular dystrophy (DMD) gene is dystrophin1, a rod-like protein2 that protects striated myocytes from contraction-induced injury3,4. Dystrophin-related protein (or utrophin) retains most of the structural and protein binding elements of dystrophin5. Importantly, normal thymic expression in DMD patients6 should protect utrophin by central immunologic tolerance. We designed a codon-optimized, synthetic transgene encoding a miniaturized utrophin (µUtro), deliverable by adeno-associated virus (AAV) vectors. Here, we show that µUtro is a highly functional, non-immunogenic substitute for dystrophin, preventing the most deleterious histological and physiological aspects of muscular dystrophy in small and large animal models. Following systemic administration of an AAV-µUtro to neonatal dystrophin-deficient mdx mice, histological and biochemical markers of myonecrosis and regeneration are completely suppressed throughout growth to adult weight. In the dystrophin-deficient golden retriever model, µUtro non-toxically prevented myonecrosis, even in the most powerful muscles. In a stringent test of immunogenicity, focal expression of µUtro in the deletional-null German shorthaired pointer model produced no evidence of cell-mediated immunity, in contrast to the robust T cell response against similarly constructed µDystrophin (µDystro). These findings support a model in which utrophin-derived therapies might be used to treat clinical dystrophin deficiency, with a favorable immunologic profile and preserved function in the face of extreme miniaturization.

Topology of the yeast Ras converting enzyme as inferred from cysteine accessibility studies.

The Ras converting enzyme (Rce1p) is an endoprotease that is involved in the post-translational processing of the Ras GTPases and other isoprenylated proteins. Its role in Ras biosynthesis marks Rce1p as an anticancer target. By assessing the chemical accessibility of cysteine residues substituted throughout the Saccharomyces cerevisiae Rce1p sequence, we have determined that yeast Rce1p has eight segments that are protected from chemical modification. Notably, the three residues that are essential for yeast Rce1p function (E156, H194, and H248) are all chemically inaccessible and associated with separate protected segments. By specifically assessing the chemical reactivity and glycosylation potential of the NH2 and COOH termini of Rce1p, we further demonstrate that Rce1p has an odd number of transmembrane spans. Substantial evidence that the most NH2-terminal segment functions as a transmembrane segment with the extreme NH2 terminus projecting into the endoplasmic reticulum (ER) lumen is presented. Because each of the remaining seven segments is too short to contain two spans and is flanked by chemically reactive positions, we infer that these segments are not transmembrane segments but rather represent compact structural features and/or hydrophobic loops that penetrate but do not fully span the bilayer (i.e., re-entrant helices). We thus propose a topological model in which yeast Rce1p contains a single transmembrane helix localized at its extreme NH2 terminus and one or more re-entrant helices and/or compact structural domains that populate the cytosolic face of the ER membrane. Lastly, we demonstrate that the natural cysteine residues of Rce1p are chemically inaccessible and fully dispensable for in vivo enzyme activity, formally eliminating the possibility of a cysteine-based enzymatic mechanism for this protease.

Alternative routes of insulin delivery.

Parenteral route of insulin administration has been the mode of treatment for all Type 1 diabetics and Type 2 diabetics with complications. Patient compliance has really been a major concern for this route of administration. Several alternative routes of administration are under consideration for effective glycemic control, including oral, inhaled, buccal, nasal, and patch routes. One of the approaches involving inhaled insulin has now reached the market. Several other candidates may reach the market in the near future, the promising one being oral insulin.

Proteolytic processing of certain CaaX motifs can occur in the absence of the Rce1p and Ste24p CaaX proteases.

The CaaX motif directs C-terminal protein modifications that include isoprenylation, proteolysis and carboxylmethylation. Proteolysis is generally believed to require either Rce1p or Ste24p. While investigating the substrate specificity of these proteases, using the yeast a-factor mating pheromone as a reporter, we observed Rce1p- and Ste24p-independent mating (RSM) when the CKQQ CaaX motif was used in lieu of the natural a-factor CVIA motif. Uncharged or negatively charged amino acid substitutions at the a(1) position of the CKQQ motif prevented RSM. Alanine substitutions at the a(2) and X positions enhanced RSM. Random mutagenesis of the CaaX motif provided evidence that RSM occurs with approximately 1% of all possible CaaX motif permutations. Combined mutational and genetic data indicate that RSM-promoting motifs have a positively charged amino acid at the a(1) position. Two of nine naturally occurring yeast CaaX motifs conforming to this pattern promoted RSM. The activity of the isoprenylcysteine carboxyl methyltransferase Ste14p was required for RSM, indicating that RSM-promoting CaaX motifs are indeed proteolysed. RSM was enhanced by the overexpression of Axl1p or Ste23p, suggesting a role for these M16A subfamily metalloproteases in this process. We have also determined that an N-terminal extension of the a-factor precursor, which is typically removed by the yeast M16A enzymes, is required for optimal RSM. These observations suggest a model that involves targeting of the a-factor precursor to the peptidosome cavity of M16A enzymes where subsequent interactions between RSM-promoting CaaX motifs and the active site of the M16A enzyme lead to proteolytic cleavage.