Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
2001-01-09
2003-10-21
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S320100, C435S325000, C435S366000, C435S368000, C435S369000, C435S370000, C530S350000, C536S023720, C536S023400
Reexamination Certificate
active
06635466
ABSTRACT:
BACKGROUND OF THE INVENTION
Gene transfer for the correction of inborn errors of metabolism and neurodegenerative diseases of the central nervous system (CNS), and for the treatment of cancer has been accomplished with recombinant adenoviral vectors. High particle doses, however, are required for efficacy in mice and rats, and for the infection of large numbers of cells in monkeys. The delivery of such high particle loads has the negative side effect of inducing an immune response in vivo. Thus, gene transfer to brain tissues with adenovirus type 2 (Ad2) or Ad5 vectors is inefficient, which is also true for endothelia, smooth muscle, and differentiated airway epithelia. Methods that improve the efficiency of adenovirus-mediated gene transfer to cells of the CNS, or other target cells such as tumor cells, could reduce the particle load required to achieve sufficient levels of transduction. Improved efficiency could then reduce toxicity and increase the therapeutic index.
There is a continuing need for vehicles and methods for efficient adenovirus-mediated gene transfer of nucleic acids or proteins to cells, such as cells of the CNS or tumor cells.
SUMMARY OF THE INVENTION
The present invention provides adenovirus serotype 30 (Ad30) fiber proteins, such as the polypeptide encoded by SEQ ID NO:1. The present invention also provides a polynucleotide encoding such Ad30 fiber protein, such as the polynucleotide encoded by SEQ ID NO:12. As used herein, the term “fiber protein” includes variants or biologically active or inactive fragments of this polypeptide. A “variant” of the polypeptide is a fiber protein that is not completely identical to a native fiber protein. A variant fiber protein can be obtained by altering the amino acid sequence by insertion, deletion or substitution of one or more amino acid. The amino acid sequence of the protein is modified, for example by substitution, to create a polypeptide having substantially the same or improved qualities as compared to the native polypeptide. The substitution may be a conserved substitution. A “conserved substitution” is a substitution of an amino acid with another amino acid having a similar side chain. A conserved substitution would be a substitution with an amino acid that makes the smallest change possible in the charge of the amino acid or size of the side chain of the amino acid (alternatively, in the size, charge or kind of chemical group within the side chain) such that the overall peptide retains its spacial conformation but has altered biological activity. For example, common conserved changes might be Asp to Glu, Asn or Gln; His to Lys, Arg or Phe; Asn to Gln, Asp or Glu and Ser to Cys, Thr or Gly. Alanine is commonly used to substitute for other amino acids. The 20 essential amino acids can be grouped as follows: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine having nonpolar side chains; glycine, serine, threonine, cystine, tyrosine, asparagine and glutamine having uncharged polar side chains; aspartate and glutamate having acidic side chains; and lysine, arginine, and histidine having basic side chains. Stryer, L.
Biochemistry
(2d edition) W. H. Freeman and Co. San Francisco (1981), p. 14-15; Lehninger, A.
Biochemistry (
2d ed., 1975), p. 73-75.
It is known that variant polypeptides can be obtained based on substituting certain amino acids for other amino acids in the polypeptide structure in order to modify or improve biological activity. For example, through substitution of alternative amino acids, small conformational changes may be conferred upon a polypeptide that result in increased bioactivity. Alternatively, amino acid substitutions in certain polypeptides may be used to provide residues that may then be linked to other molecules to provide peptide-molecule conjugates that retain sufficient properties of the starting polypeptide to be useful for other purposes.
One can use the hydropathic index of amino acids in conferring interactive biological function on a polypeptide, wherein it is found that certain amino acids may be substituted for other amino acids having similar hydropathic indices and still retain a similar biological activity. Alternatively, substitution of like amino acids may be made on the basis of hydrophilicity, particularly where the biological function desired in the polypeptide to be generated in intended for use in immunological embodiments. The greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity. U.S. Pat. No. 4,554,101. Accordingly, it is noted that substitutions can be made based on the hydrophilicity assigned to each amino acid. In using either the hydrophilicity index or hydropathic index, which assigns values to each amino acid, it is preferred to conduct substitutions of amino acids where these values are ±2, with ±1 being particularly preferred, and those with in ±0.5 being the most preferred substitutions.
The variant amino acid molecule of the present invention has at least 50%, at least about 80%, or even at least about 90% but less than 100%, contiguous amino acid sequence homology or identity to the amino acid sequence of a corresponding native nucleic acid molecule or polypeptide.
The amino acid sequence of the variant fiber protein corresponds essentially to the native fiber protein's amino acid sequence. As used herein “corresponds essentially to” refers to a polypeptide sequence that will elicit a biological response substantially the same as the response generated by native fiber protein. Such a response may be at least 60% of the level generated by native fiber protein, and may even be at least 80% of the level generated by native fiber protein.
A variant of the invention may include amino acid residues not present in the corresponding native fiber protein, or may include deletions relative to the corresponding native fiber protein. A variant may also be a truncated “fragment” as compared to the corresponding native fiber protein, i.e., only a portion of a full-length protein. For, example, the polypeptide of the present invention may contain one or more of the three regions of an Ad30 fiber, i.e., a tail region (such as amino acids 1-45 of SEQ ID NO:1), a shaft region (such as amino acids 46-188 of SEQ ID NO:1) or a knob region (such as amino acids 189-371 of SEQ ID NO:1). Fiber protein variants also include peptides having at least one D-amino acid.
The variant fiber protein of the present invention may be expressed from an isolated DNA sequence encoding the variant fiber protein. The amino acid changes from the native to the variant fiber protein are achieved by changing the codons of the corresponding nucleic acid sequence. “Recombinant” is defined as a peptide or nucleic acid produced by the processes of genetic engineering. It should be noted that it is well-known in the art that, due to the redundancy in the genetic code, individual nucleotides can be readily exchanged in a codon, and still result in an identical amino acid sequence. The terms “protein,” “peptide” and “polypeptide” are used interchangeably herein.
The Ad30 fiber protein as described above may be operably linked to an amino acid sequence for a therapeutic agent. An amino acid or nucleic acid is “operably linked” when it is placed into a functional relationship with another amino acid or nucleic acid sequence. For example, DNA a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the amino acid or nucleic acid sequences being linked are contiguous, and, in the case of a secretory leader in DNA, contiguous and in reading phase. However, enhancers do no
Davidson Beverly L.
Law Lane K.
Mosher Mary E.
Schwegman Lundberg Woessner & Kluth P.A.
University of Iowa Research Foundation
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