Method and vector for producing and transferring...

Details Method and vector for producing and transferring... Method and vector for producing and transferring...

2000-10-13

2003-04-08

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S069100, C435S069700, C435S091400, C435S320100, C435S337000, C435S252330, C530S333000, C530S343000, C536S023100, C536S024100

Reexamination Certificate

active

06544786

ABSTRACT:

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not applicable.
BACKGROUND
1. Technical Field
Disclosed are methods for protein trans-splicing, use of trans-spliced proteins in gene therapies and gene therapy vectors that encode proteins that trans-splice. In particular, a method for trans-splicing dystrophin and use in gene therapies of recombinant Adeno-Associated Virus (rAAV) particles that encode trans-spliced dystrophin.
2. Description of the Related Art
Protein splicing elements, protein introns, were first discovered in yeast (Kane, P. M., Yamashiro, C. T., Wolczyk, D. F., Neff, N., Goebl, M., and Stevens, T. H. (1990), Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)−adenosine triphosphatase,
Science
250, 651-657, incorporated herein by reference). Four years later, after six more protein introns had been characterized, they were renamed “inteins” (Perler, F. B., Davis, E. O., Dean, G. E., Gimble, F. S., Jack, W. E., Neff, N., Noren, C. J., Thorner, J., and Belfort, M. (1994), Protein splicing elements: inteins and exteins—a definition of terms and recommended nomenclature,
Nucleic Acids Res.
22, 1125-1127, incorporated herein by reference). Over 100 inteins have since been found in various precursor (host) proteins in a variety of bacterial, archaebacterial and eukaryotic organisms. An intein is defined as a protein sequence which is embedded in-frame within a precursor protein sequence and which is excised during a maturation process called protein splicing. Protein splicing is a post-translational event involving precise excision of the intein sequence and concomitant ligation of the flanking sequences (N- and C-exteins) by a normal peptide bond.
The chemical mechanism of protein splicing was proposed by several groups in 1993 (Wallace, C. J. (1993), The curious case of protein splicing: mechanistic insights suggested by protein semisynthesis,
Protein Sci.
2, 697-705; Cooper, A. A., Chen, Y. J., Lindorfer, M. A., and Stevens, T. H. (1993), Protein splicing of the yeast TFP1 intervening protein sequence: a model for self-excision,
EMBO J.
12, 2575-2583; Xu, M.-Q., Southworth, M. W., Mersha, F. B., Hornstra, L. J., and Perler, F. B. (1993), In vitro protein splicing of purified precursor and the identification of a branched intermediate,
Cell
75, 1371-1377, both of which are incorporated herein by reference) and has since been supported and refined by experimental data (Xu, M.-Q., Comb, D. G., Paulus, H., Noren, C. J., Shao, Y., and Perler, F. B. (1994), Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation,
EMBO J.
13, 5517-5522; Xu, M.-Q. and Perler, F. B, (1996), The mechanism of protein splicing and its modulation by mutation,
EMBO J.
15, 5146-5153; both of which are incorporated herein by reference). Briefly, a typical intein folds upon itself, bringing the upstream and downstream splice junctions together to form an active center. Splicing involves an N—S or an N—O acyl shift at the splice sites, formation of a branched intermediate, and cyclization of an invariant Asn residue at the C-terminus of the intein to form succinimide, leading to excision of the intein and ligation of the exteins. Amino acid residues that participate directly in the splicing reaction include a nucleophilic amino acid (Cys or Ser), both at the beginning of the intein sequence and at the beginning of the C-extein sequence (Cys, Ser, or Thr), an internal His, and an Asn at the end of the intein sequence. Practical uses of inteins have been made by engineering controllable inteins which undergo controllable protein splicing or cleavage in vitro, including protein trans-splicing by intein fragment reassembly in vitro (Southworth, M. W., Adam, E., Panne, D., Byer, R., Kautz, R., and Perler, F. B. (1998), Control of protein splicing by intein fragment reassembly,
EMBO J.
17, 918-926; Mills, K. V., Lew, B. M., Jiang, S.-Q., and Paulus, H. (1998), Protein splicing in trans by purified N- and C-terminal fragments of the
Mycobacterium tuberculosis
RecA intein,
Proc. Natl. Acad. Sci. U.S.A.
95, 3543-3548; both of which are incorporated herein by reference). In vivo protein trans-splicing has also been shown through intein engineering (Shingledecker, K., Jiang, S.-Q., and Paulus, H. (1998), Molecular dissection of the
Mycobacterium tuberculosis
RecA intein: design of a minimal intein of a trans-splicing system involving two intein fragments,
Gene
207, 187-195; Wu, H., Xu, M.-Q., and Liu, X.-Q (1998b), Protein trans-splicing and functional mini-inteins of a cyanobacterial DnaB intein,
Biochim. Biophys. Acta
1387, 422-432; each of which are incorporated herein by reference) and the discovery of a naturally occurring trans-splicing intein (Wu, H., Hu, Z., and Liu, X.-Q. (1998a), Protein trans-splicing by a split intein encoded in a split DnaE gene of
synechocystis
sp. PCC6803,
Proc. Natl. Acad. Sci. U.S.A.
95, 9226-9231; incorporated herein by reference). But until now, no practical use has been described for spontaneous or automatic in vivo protein trans-splicing.
Duchenne muscular dystrophy (DMD) is the most common form of X-linked muscular dystrophy, with a world-wide incidence of one in 3,500 male births (Emery, A. E. H., Duchenne Muscular Dystrophy, Oxford University Press, 1993: 392; incorporated herein by reference). DMD patients appear normal until 3-5 years of age, when they begin to experience progressive muscular weakness, starting with large proximal skeletal muscles. The typical affected individual is wheelchair-bound by the age of 12 and succumbs to cardiac or respiratory failure in the mid to late 20s. Becker muscular dystrophy (BMD) is a milder form with delayed onset and longer life span. Most DMD/BMD cases are transmitted via an unaffected mother (heterozygote), whereas 30% of cases have no previous family history and are considered to be due to a de novo mutation in the germ line of either the mother or her parents.
DMD and BMD are caused by a defective dystrophin protein in a patient's muscle cells (See, Straub, V. and Campbell, K. P. (1997), Muscular dystrophies and the dystrophin-glycoprotein complex,
Curr. Opin. Neurol.
10, 168-175; Brown, Jr., R. H. (1997), Dystrophin-associated proteins and the muscular dystrophies,
Ann. Rev. Med.
48, 457-466; Michalak, M. and Opas, M. (1997), Functions of dystrophin and dystrophin associated proteins,
Curr. Opin. Neurol.
10, 436-442, for recent reviews; each of which are incorporated herein by reference). Dystrophin is a large protein of 3,685 aa and has three structurally distinct regions. The N-terminal region is 136 aa long and forms a globular domain. The C-terminal region is 645 aa long and forms a second globular domain. The central region is a long and rod-like domain that consists of 24 repeats of a triple helical coiled-coil, or of 9 repeats in the smaller, but still functional, Becker form. The N- and C-terminal domains are separated, both in primary sequence and in tertiary structure by the central region. Each repeat is approximately 109 aa long, and there is 10-25% sequence identity between repeats. Each individual repeat is believed to fold independently into a structural module, and neighboring repeats are connected by a short, flexible linker sequence.
Dystrophin is a part of the dystrophin-glycoprotein complex and is thought to function by forming a submembrane lattice which enhances the tensile strength of the muscle membrane and by serving as an anchor for membrane proteins. The human dystrophin gene was identified in 1986 (Monaco, A. P., Neve, R. L., Colletti-Feener, C., Bertelson, C. J., Kurnit, D. M., and Kunkel, L. M. (1986), Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene,
Nature
323, 646-650; incorporated herein by reference), the dystrophin protein was identified in 1987 (Hoffman, E. P., Brown, Jr., R. H., and Kunkel, L. M. (1987), Dystrophin: the protein product of the Duchenne muscular dystrophy locus,
Cell
51, 919

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