The present invention provides replication-defective hybrid retroviral vectors comprising GaLV components and methods for preparing and using such vectors. The vectors comprise a envelope component, a core component and a defective genome, at least one of which is derived from GaLV. The vectors can comprise the minimal cis acting sequences from GaLV that allow packaging of the defective genome in a hybrid virion.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT 
Not applicable. 
BACKGROUND OF THE INVENTION 
The present invention relates generally to retroviral vectors. In 
particular, the invention relates to retroviral vectors comprising nucleic 
acid sequences from Gibbon Ape Leukemia Virus. 
Considerable effort is now being directed to introducing engineered genes 
into mammalian cells for a variety of applications including gene therapy 
and the production of transgenic animals. Such strategies are dependent 
upon the development of effective means for safe delivery of genes to 
appropriate target cells and tissues. 
Retroviral vectors are particularly useful for directing desired 
polynucleotides to the appropriate cells and integration of the 
polynucleotides in the host cell genome. For example, the majority of the 
approved gene transfer trials in the United States rely on 
replication-defective retroviral vectors harboring a therapeutic 
polynucleotide sequence as part of the retroviral genome (Miller et al. 
Mol. Cell. Biol. 10:4239 (1990); Kolberg R J. NIH Res. 4:43 (1992); 
Cornetta et al. Hum. Gene Ther. 2:215 (1991)). As is known in the art, the 
major advantages of retroviral vectors for gene therapy are the high 
efficiency of gene transfer into certain types of replicating cells, the 
precise integration of the transferred genes into cellular DNA, and the 
lack of further spread of the sequences after gene transfer. 
Unfortunately, many human cells are not efficiently infected by prior art 
retroviral vectors. Reduced susceptibility to retroviral infection is most 
likely due to inefficiencies in one of three stages of viral replication: 
1) binding to retroviral receptors on the cell surface and early viral 
entry, 2) late entry and transport of the viral genome to the cell nucleus 
and integration of the viral genome into the target cell DNA, and 3) 
expression of the viral genome. These three stages are governed, 
respectively, by the viral envelope proteins, the viral core proteins, and 
the viral genome. All three of these components must function efficiently 
in a target cell to achieve optimal therapeutic gene delivery. 
Gibbon Ape Leukemia Virus (GaLV) uses a cell surface internalization 
receptor that is different from those of the available retroviral vectors 
and thus allows infection of cells and tissues normally resistant to 
retroviral infection. The human receptor for GaLV has recently been cloned 
and shows a wide cell type and species distribution. Johann et al., J. 
Virol. 66:1635-1640 (1992). Indeed, GaLV can infect many mammalian species 
with the notable exception of mouse cells. The same receptor is used by 
simian sarcoma associated virus (SSAV), a strain of GaLV. Sommerfelt et 
al., Virol. 176:58-59 (1990). 
The construction of hybrid virions having GaLV envelope proteins has been 
demonstrated. For instance, Wilson et al., J. Virol. 63:2374-2378 (1989), 
describe preparation of infectious hybrid virions with GaLV and human 
T-cell leukemia virus retroviral env glycoproteins and the gag and pol 
proteins of the Moloney murine leukemia virus (MoMLV). In addition, Miller 
et al., J. Virol. 65:2220-2224 (1991), describe construction of hybrid 
packaging cell lines that express GaLV envelope and MoMLV gag-pol 
proteins. 
Existent retroviral vectors capable of infecting human cells all contain 
core and genome components that derive from MoMLV. For human cells which 
are resistant to efficient infection by such vectors at any of the three 
stages noted above, new vectors comprising improved envelope, core or 
regulatory sequences must be designed. Thus, there is a need to design 
retroviral vectors components which can be used to introduce genes into 
human cells not efficiently infected by the currently utilized retroviral 
vectors. The present invention addresses these and other needs. 
SUMMARY OF THE INVENTION 
The present invention provides recombinant DNA constructs comprising a 
defective viral genome having a polynucleotide sequence of interest and a 
GaLV component. For instance, the GaLV component may be a GaLV packaging 
site which directs packaging of the defective viral genome in an 
infectious, replication-defective virion. The packaging site typically 
consists of between about 150 base pairs and about 1500 base pairs and 
includes a sequence extending from about position 200 to about position 
1290 of the sequence shown in SEQ ID NO.:1. 
The construct may further comprise GaLV regulatory sequences which direct 
expression of the polynucleotide of interest. Typically, the regulatory 
sequences comprise a GaLV (e.g., GaLV SEATO or GaLV SF) 5' or 3' LTR 
promoter. 
The invention also relates to mammalian cells comprising the defective 
viral genome described above. The mammalian cells may be packaging cells, 
in which case the cells will also contain retroviral gag, pol and env 
genes. These genes may be derived from MoMLV, GaLV SF or GaLV SEATO. 
Packaging cells conveniently used in the invention include PG13 and 17. 
The invention further provides isolated hybrid virions comprising GaLV 
(e.g., SF or SEATO) envelope proteins and an RNA genome comprising a 
polynucleotide sequence of interest and a GaLV component. The virions 
typically contain GaLV core proteins. MoMLV core proteins can also be 
used. 
The invention also provides isolated recombinant DNA constructs comprising 
polynucleotide sequences which encode an infectious GaLV virion capable of 
infecting a mammalian cell and producing functional viral progeny. The 
infectious clones typically comprise about 97% GaLV SEATO sequences and 3% 
GaLV SF sequences. 
Also disclosed are methods of introducing a polynucleotide of interest into 
human cells using the hybrid virions described above. The methods are 
preferably used as part of a gene therapy protocol for treating a human 
patient. 
DEFINITIONS 
A "hybrid virion" is a virion comprising genome, core, and envelope 
components derived from more than one virus. The term specifically 
includes "pseudovirions" which historically have been defined as 
containing the genome from one virus and the structural proteins from 
another. 
A "packaging cell" is a genetically constructed mammalian tissue culture 
cell that produces the necessary viral structural proteins required for 
packaging. The cells are incapable of producing infectious virions until a 
defective genome is introduced into the cells. The genetic material for 
the viral structural proteins is not transferred with the virions produced 
by the cells, hence the virus cannot replicate. 
A "replication-defective" virion or retroviral vector is one produced by a 
packaging cell as defined above. Such a virion infects a target cell but 
is incapable of producing progeny virions which can infect other cells. 
Two polynucleotides or polypeptides are said to be "identical" if the 
sequence of nucleotides or amino acid residues in the two sequences is the 
same when aligned for maximum correspondence. Optimal alignment of 
sequences for comparison may be conducted by the local homology algorithm 
of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology 
alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), 
by the search for similarity method of Pearson and Lipman Proc. Natl. 
Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of 
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin 
Genetics Software Package, Genetics Computer Group, 575 Science Dr., 
Madison, Wis.), or by inspection. These references are incorporated herein 
by reference. 
The percentage of sequence identity between two sequences is determined by 
comparing two optimally aligned sequences over a window of comparison of 
at least 20 positions. The percentage is calculated by determining the 
number of positions at which the identical nucleic acid base or amino acid 
residue occurs in both sequences to yield the number of matched positions, 
dividing the number of matched positions by the total number of positions 
in the window of comparison (i.e., the window size), and multiplying the 
result by 100 to yield the percentage of sequence identity. 
For instance, a preferred method for comparing sequences uses the GAP 
program based on the algorithm of Needleman at al., supra. Typically, the 
default values for all parameters are selected. These are gap weight: 5.0, 
length weight: 0.30, average match: 1.0, and average mismatch: 0.0. 
The term "substantial identity" means that a polynucleotide or polypeptide 
comprises a sequence that has at least 80% sequence identity, preferably 
90%, more preferably 95% or more, compared to a reference sequence over a 
comparison window of about 20 bp to about 2000 bp, typically about 50 to 
about 1500 bp, usually about 350 bp to about 1200. The values of percent 
identity are determined using the GAP program, above. 
Another indication that nucleotide sequences are substantially identical is 
if two molecules hybridize to each other under stringent conditions. 
Stringent conditions are sequence dependent and will be different in 
different circumstances. Generally, stringent conditions are selected to 
be about 5.degree. C. lower than the thermal melting point (Tm) for the 
specific sequence at a defined ionic strength and pH. The Tm is the 
temperature (under defined ionic strength and pH) at which 50% of the 
target sequence hybridizes to a perfectly matched probe. Typically, 
stringent conditions will be those in which the salt concentration is 
about 0.2 molar at pH 7 and the temperature is at least about 60.degree. C 
.