Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-05-20
2002-09-10
Crouch, Deborah (Department: 1633)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C435S235100
Reexamination Certificate
active
06448390
ABSTRACT:
FIELD OF THE INVENTION
This invention includes retrovirus envelope mutants into which heterologous peptide or glycopeptide sequences can be fused for expression and stable presentation on retroviral, viral and liposome vectors. The invention further relates to methods of making and using these retrovirus envelopes for gene and drug therapy.
I. BACKGROUND
1. Gene Therapy Vectors
Numerous gene therapy vectors have been created. These vectors are constantly being engineered to overcome problems caused by tropism, infectivity and virus stability. Engineered gene therapy virus vectors include adenoviruses (see as examples, D. Armentano et al., 1998 U.S. Pat. No. 5,707,618; T. J. Wickham et al., 1998 U.S. Pat. No. 5,731,190; and M. Cotten et al., 1997 U.S. Pat. No. 5,693,509), herpes simplex viruses (R. L. Martuza et al., 1998 U.S. Pat. No. 5,728,379) and retrovirus vectors. Non-viral vectors include episomal and liposomal vectors such as those described in M. J. Cooper, 1997 U.S. Pat. No. 5,624,820; and L. Li et al., 1997 U.S. Pat. No. 5,641,508.
Retroviruses have been preferred vectors for gene therapy based on their ability to integrate retroviral DNA into the genome of the host cell. Retroviral gene therapy patents include vectors with multiple cloning sites (M. Eglitis et al., 1997 U.S. Pat. No. 5,672,510), retroviruses with mixed LTRs (H. M. Temin et al., 1996 U.S. Pat. No. 5,554,524), retroviruses that deliver genetic elements that stimulate an immune response (H. E. Gruber et al., 1998 U.S. Pat. Nos. 5,716,826 and 5,716,426), and vectors with specific envelope proteins (E. F. Vanin et al., 1998 U.S. Pat. No. 5,710,037).
2. Retroviral Envelope Mutations and Gene Therapy
The retrovirus envelope protein is the viral element that allows a gene therapy vector to recognize and bind to the target, typically a host cell. The native or wild-type retrovirus envelope protein has a natural tropism for certain target cells, which typically must be overcome if the virus is to be used as a gene therapy vector. However, the envelope protein engineered to overcome wild type tropisms must nevertheless maintain the characteristics of: (1) surface (SU) protein stability such that it is not shed from the virus particle too quickly; and (2) infectivity wherein the virion can infect the host cell and introduce the genetic material it is carrying.
The envelope protein in all retroviruses is produced as a glycoprotein precursor that matures into two cleavage products: the surface protein (SU) and transmembrane protein (TM). TM and SU are held together by disulfide bonds and perhaps other non-covalent interactions (J. N. Coffin et al., 1997
Retroviruses
Cold Spring Harbor Press). Envelope shedding, in Moloney murine leukemia virus (MoMLV) for example, occurs as result of the weak linkage created by the disulfide bonds between the TM (p15E) and the SU (gp70). Although mutations have been made to prevent cleavage of the envelope precursor protein in MoMLV gp80 into SU and TM, typically there has been an associated loss of incorporation into virions observed with these mutants making them undesirable vectors (E. O. Freed et al., 1987
J. Virol.
61: 2852-6). Finally, in HIV, the endoproteolytic cleavage of the envelope precursor protein has been demonstrated to be required for the activation of HIV (J. M. McCune et al., 1988
Cell
53: 55-67).
Envelope proteins also are known to possess highly conserved functional domains. In MoMLV, amino acid residues 1-33 constitute the leader sequence; amino acids 34-263 constitute the receptor binding domain; amino acids 264-312 comprise the hinge region; and residues 313-469 constitute the body portion of the surface protein (J. M. Mason et al., 1997 U.S. Pat. No. 6,643,770). Mutagenesis analysis of the envelope protein has led to the discovery of other amino acid residues that appear responsible for receptor binding (A. J. MacKrell et al., 1996
J. Virol.
70: 1768-74) and fusion events (Y. Bae et al., 1997
J. Virol.
71: 2092-9). Although at least one group has proposed that the N-terminal 72 residues of the amphotropic 4070A isolate are not required, for amphotropic receptor usage (C. Peredo et al., 1996
J. Virol.
70: 3142-52), other researchers have demonstrated that the N-terminus of the envelope protein is required especially when preparing fusion envelope proteins (see for examples, F-L. Cosset et al., 1995
J. Virol.
69: 6314-22; S. Valsesia-Wittmann et al., 1996
J. Virol.
70: 2059-64; and J. M. Heard et al., 1991
J. Virol.
65: 4026-32). Additional mutagenic analysis of retrovirus (e.g., in PVC-211 murine leukemia virus and MoMLV) envelope proteins has been described and is discussed in the following: M. Masuda et al., 1996
J. Virol.
70: 8534-9; and A. J. MacKrell et al., 1996; and H. Skov et al., 1993
J. Gen. Virol.
74:707-14).
3. Fusion Glycoproteins
One method of overcoming the retrovirus' natural tropism is by expressing an envelope fusion glycoprotein. A fusion glycoprotein contains the retroviral envelope (env) protein, e.g., the SU protein, linked to a selected peptide or glycopeptide. For examples, see R. W. Paul et al., 1998 U.S. Pat. No. 5,736,387 and S. J. Russell et al., 1998 U.S. Pat. No. 5,723,287. Many of the engineered envelope proteins created to target the retrovirus particle to other cells comprise insertions of heterologous peptides into the amino terminus of SU (F-L Cosset et al., 1995
J. Virol.
69: 6314-22). Fusion glycoproteins have been developed to compensate for the folding problems created due to changes in the glycosylated pattern. (S. Kayman et al., 1997 U.S. Pat. No. 5,643,756).
SUMMARY OF THE INVENTION
The inventors disclose novel mutant envelope proteins which when linked or fused to a heterologous peptide or glycopeptide have enhanced stability and maintain retrovirus virion titer and infectivity levels comparable to that observed for wild type retrovirus envelope proteins. Due to the ability to restore the target penetration capability that is lost or greatly diminished upon fusion of a heterologous sequence into the envelope protein, vectors containing these mutant envelope proteins have an increased ability to penetrate targets, typically cells, and a correspondingly increased ability to deliver nucleic acids or drugs. Further, due to the increased association between SU and TM the mutant envelope proteins are more stable and vectors containing these mutant envelope proteins are less likely to shed off of the vector or infective particle. Decreased envelope shedding increases the life span of a virion so it can survive such mechanical stressors as freezing and thawing or vascular shearing forces. Correspondingly, vectors containing these mutant envelope proteins would be extremely useful as nucleic acid and drug delivery vehicles. Methods of identifying retrovirus mutant envelope proteins possessing these desirable characteristics based on three-dimensional structural motifs are also disclosed.
This invention discloses isolated nucleic acid molecules encoding retrovirus envelope proteins or polypeptide fragments thereof having decreased shedding of binding sequences through the suppression of envelope protein cleavage comprising an amino acid substitution in at least one amino acid of at least one of seven motifs or corresponding amino acid residues in other retrovirus envelope proteins. The first five (5) motifs as described for MoMuLV which exhibit this function comprise the following amino acids: (1)
104
Lys,
107
Glu,
90
Thr,
102
Arg and
108
Thr; (2)
124
Arg,
138
Tyr,
128
Ser,
132
Gly,
134
Pro,
121
Gly, and
133
Gly; (3)
223
Arg,
225
Arg,
224
Leu,
16
Glu,
24
Thr, and
201
Thr; (4)
137
Phe,
135
Asp,
136
Ser,
208
Arg, and
217
Gly; and (5)
142
Trp,
152
Trp,
210
Tyr,
141
Tyr and
151
Tyr. For motif 6, which comprises
227
Gln,
228
Asn, and
243
Asp, the isolated nucleic acid molecule encodes a retrovirus envelope protein or polypeptide fragment thereof having increased penetration capability through restoration of the function of residue 8His and decreased shedding of binding
Albritton Lorraine Moore
Zavorotinskaya Tatiana
Crouch Deborah
Morgan & Lewis & Bockius, LLP
Sorbello Eleanor
The University of Tennessee Research Corporation
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