JP2002533128A - Human fibroblast growth factor receptor 1 as a co-receptor for infection by adeno-associated virus 2 - Google Patents

Abstract

  (57) [Summary] The present invention relates generally to the field of gene therapy. More particularly, it relates to gene transfer using adeno-associated virus, and methods of increasing transcription and promoting transgene replication. The present invention shows that AAV requires human fibroblast growth factor receptor 1 (FGFR 1) as a coreceptor for successful entry into host cells. Methods and compositions for exploiting this discovery in AAV vector-mediated gene therapy are disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION [0001] This application is filed with US Provisional Patent Application No. 60 / 114,596 (December 31, 1998).
Claims the benefits of National Institutes of
Health (Centers of Excellence in Mole)
Public Health Se from the Central Hematology)
rvic transfer numbers HL-48342, HL-53586, HL-58881,
And in the present invention in accordance with DK-49218, the government may have rights.

1. Field of the Invention The present invention relates generally to the field of gene therapy. More specifically, the present invention relates to gene transfer using adeno-associated virus and methods for increasing transgene transcription and promoting replication.

2. Description of the Related Art Gene therapy protocols involving recombinant viral vectors have received widespread attention as new weapons against disease. Retrovirus-based and adenovirus-based vector systems of different viral vectors used for gene transfer have been most extensively studied. In recent years, adeno-associated virus (AAV)
Emerging as a strong alternative to the more commonly used retroviral and adenoviral vectors (Muzyczka, 1992; Carter, 19
92; Flotte and Carter, 1995; Chatterjee et al.
1995; Chatterjee and Wong, 1996). Although studies using retroviral-mediated gene transfer and adenovirus-mediated gene transfer have been linked to previous potential oncogenic properties and later to related immunogenicity issues, AAV has shown that any such (Berns and Bohenzky, 1987; Berns and Giraud, 1996).

The viral genome of AAV is a single-stranded DNA of 4,680 nucleotides, flanked on both ends by a 145 nucleotide long palindromic inverted terminal repeat (ITR) (Srivastava et al., 1983). Wild-type AAV has been shown to integrate into the human chromosome in a site-specific manner (Kotin et al., 1991; S
amulski et al., 1991), whereas recombinant AAV vectors do not appear to integrate site-specifically (Kearns et al., 1996; Ponazha).
gan et al., 1997). In previous studies, the transduction efficiency of AAV vectors in permissive cells was shown to be a single-chain D sequence-binding protein (ssD-BP) that preferentially interacts with the D (-) sequence within the AAV ITR sequence. It has been demonstrated to be highly related to the phosphorylation state of proteins (Qing et al., 1997; Qi
ng et al., 1998; and US Patent Application Serial No. 09 / 145,379, which is specifically incorporated herein by reference in its entirety). Two independent groups were rate-limited for efficient transduction by AAV following infection (rate-li).
The evidence suggests that the process is a viral double-stranded DNA synthesis (Fisher et al., 1996; Ferrari et al., 1996). Furthermore, the tyrosine phosphorylation status of ssD-BP is similar to that of AAV in vivo.
It proved to be highly related to the efficiency of mediated transgene expression.

AAV possesses a broad host range that crosses species boundaries (Muzyczka, 1
992). Recently, cell surface heparan sulfate proteoglycan (HSPG) has been
V receptor (Summerford and Samulsk)
i, 1998). The ubiquitous expression of HSPGs is probably responsible for the wide host range of AAV. However, principally, effective viral infection of the host cell involves at least two steps: attachment and entry (possibly each of at least two different cell surface macromolecules (receptors and co-receptors)).
Is also becoming increasingly clear.
A compelling example of such an event is shown in connection with infection by human immunodeficiency virus 1 (HIV1), which utilizes the cell surface CD4 antigen as the site of attachment, followed by one chemokine receptor for virus entry. (Alkhatib et al., 1996; He et al., 1997). A similar scenario was also revealed for efficient infection by adenovirus and herpes virus (Laquerere).
Bergelson et al., 1997; Montgomery et al., 1;
996; Geragty et al., 1998).

[0006] Improved efficiency of AAV infection increases the use of this vector in gene therapy applications. However, to date, the identification of receptors that successfully mediate infection of cells by AAV remains elusive. Once such receptors or factors are elucidated, it is possible to increase the efficiency of AAV-mediated gene therapy.

SUMMARY OF THE INVENTION The present invention describes for the first time a co-receptor required for AAV to enter cells. In certain embodiments of the present invention, a method is described for increasing AAV infection of a cell, comprising increasing the amount of fibroblast growth factor receptor (FGFR) on the surface of the cell, wherein the increased FGFR is Increases AAV uptake by cells. By increasing the amount of FGFR in the cell, the invention relates to any method for efficiently increasing the number of accessible AAV binding sites on the cell surface. Thus, such sites may be manipulated through the steps being manipulated or AA
It was discussed that the V-binding site may be made available through the application of factors that allow the protein conformational change to be exposed to the displayed AAV.

In certain embodiments, the increased amount of FGFR on the cell causes the cell to express FGF
This may include providing an expression construct comprising a polynucleotide encoding the R polypeptide and a promoter active in eukaryotic cells, wherein the polynucleotide is operably linked to the promoter. In a preferred embodiment, the FGFR polypeptide is FGFR1, FGFR2, FGFR3 or FGFR.
Selected from the group consisting of FR4. In another preferred embodiment, it is contemplated that the method further comprises increasing the amount of cell surface heparan sulfate proteoglycan (HSPG) on the cell. In certain embodiments, the HSP of the cell
Increasing G provides the cell with an expression construct comprising a polynucleotide encoding the HSPG polypeptide and a promoter active in eukaryotic cells, wherein the polynucleotide is operably linked to the promoter. Including. In still other embodiments, increasing the AAV binding capacity of the HSPG receptor may be possible by altering its conformation and / or glycosylation pattern.

[0009] In certain embodiments, the method may further comprise contacting the cell with an AAV vector. In a more specific embodiment, the AAV vector is a vector comprising an expression cassette comprising a selected polynucleotide and a promoter active in eukaryotic cells, wherein the polynucleotide is operably linked to the promoter. It is. More specifically, it is contemplated that the selected polynucleotide encodes a polypeptide. In other embodiments, the selected polynucleotide may encode an antisense construct. In yet another embodiment, the selected polynucleotide encodes a ribozyme. In defined embodiments, the promoter can be any promoter known to be useful in certain gene delivery applications. In certain embodiments, a promoter can be an inducible promoter. In certain preferred embodiments, the promoter is a CMV IE promoter, an SV40 IE promoter,
HSV tk promoter, β actin promoter, human globin α promoter, human globin β promoter, human globin γ promoter, RSV promoter, B19p6 promoter, α-1 antitrypsin promoter,
PGK promoter, tetracycline promoter, MMTV promoter or albumin promoter.

[0010] In certain embodiments of the invention, the expression cassette may further include a polyadenylation signal. More specifically, the polyadenylation signal can be an AAV polyadenylation signal, an SV40 polyadenylation signal or a BGH polyadenylation signal. Of course, it is understood that these are merely exemplary polyadenylation signals and that those skilled in the art can substitute for other polyadenylation signals and thus arrive at an expression cassette that can be used in the methods of the invention.

[0011] In certain embodiments, the selected polypeptide is a hormone, a tumor suppressor, an inhibitor of apoptosis, a toxin, a lymphokine, a growth factor, an enzyme, D
It can be an NA binding protein or a single chain antibody. In those embodiments where the invention provides a receptor encoding the expression construct, the expression construct may be a viral vector. More specifically, the viral vector may be selected from the group consisting of retrovirus, adenovirus, vaccinia virus, herpes virus and adeno-associated virus.

[0012] The present invention further provides a method of expressing a polynucleotide selected from an adeno-associated virus (AAV) vector in a host cell, the method comprising selecting the polynucleotide and a promoter active in a eukaryotic cell. Providing an AAV vector comprising an expression cassette comprising the polynucleotide (where the selected polynucleotide is operably linked to a promoter); and subjecting the vector to conditions that allow for uptake of the vector by a host cell. Contacting with a host cell;
And increasing the amount of fibroblast growth factor receptor (FGFR) on the surface of the cell; wherein the increased FGFR increases AAV uptake by the cell. This increased uptake of AAV is thereby caused by FGFR
Increases the level of transcription of the selected polynucleotide, which is increased relative to the transcription of the selected polynucleotide in cells where activation is not increased. Similarly, A
By providing cells with increased capacity for AV infection, the methods of the invention increase the efficiency of AAV-mediated transduction of a selected polynucleotide in a host cell.

[0013] In certain embodiments, the method of increasing FGFR on the surface of a cell comprises FGFR
Providing an expression construct comprising a polynucleotide encoding the FR polypeptide and a promoter active in a eukaryotic cell, wherein the polynucleotide is operably linked to the promoter. In a preferred embodiment, the FGFR polypeptide is FGFR1, FGFR2, FGFR3 or FGFR.
Selected from the group consisting of FR4. The method can further include increasing the amount of cell surface heparan sulfate proteoglycan (HSPG) in the cell. More specifically, the increased HSPG of the cell can be linked to a polynucleotide encoding the HSPG polypeptide and a promoter active in eukaryotic cells, wherein the polynucleotide is operably linked to the promoter. Comprising providing an expression construct comprising: In certain embodiments, the polynucleotide encoding HSPG and the polynucleotide encoding FGFR are in the same expression construct. In another embodiment, the polynucleotide encoding HSPG and the polynucleotide encoding FGFR are separated by an IRES. Each of the polynucleotide encoding HSPG and the polynucleotide encoding FGFR may be under the control of a separate promoter operable in eukaryotic cells.

[0014] It is contemplated that the methods of the present invention can be performed in any cell that is subject to gene therapy and / or delivery procedures. In certain embodiments, the host cells are red blood cells. In other defined embodiments, the red blood cells are human red blood cells. In certain embodiments, the host cells can be selected from the group consisting of bone marrow cells, peripheral blood cells, lung cells, gastrointestinal cells, endothelial cells, and cardiomyocytes. In certain embodiments, the host cell is an animal.

[0015] In certain embodiments, the method may further comprise inhibiting the function of the D sequence binding protein (D-BP) in the host cell. More specifically, the inhibition is
This may include reducing the expression of D-BP in the host cell. In other embodiments, reduced expression of D-BP can be achieved by contacting a host cell with an antisense D-BP polynucleotide. More specifically, Antisense D
-BP polynucleotide may target the translation initiation site. In other embodiments, the antisense D-BP polynucleotide targets a splice site.
In certain embodiments, the agent that decreases D-BP expression is an antibody or a small molecule inhibitor. In certain embodiments, the antibodies can be single chain antibodies or monoclonal antibodies. In other embodiments, the inhibiting comprises reducing D-BP D sequence binding activity in the host cell. In certain embodiments,
Reducing binding activity is achieved by inhibiting tyrosine phosphorylation of D-BP. More specifically, inhibiting phosphorylation is achieved by contacting a host cell with a D-BP peptide containing a tyrosine residue. In certain embodiments, inhibiting phosphorylation is achieved by contacting the host cell with an agent that inhibits tyrosine kinase. In a defined embodiment, the tyrosine kinase is an EGF-R tyrosine kinase. In certain embodiments, the factor is an inhibitor of EGF-R that reduces EGF-R protein kinase expression. In another embodiment, the inhibitor of EGF-R protein kinase is an agent that binds and inactivates EGF-R protein kinase. In yet a further embodiment, the inhibitor of EGF-R protein kinase inhibits the interaction between EGF-R and D-BP. In certain aspects, the EGF
This factor that reduces the expression of -R protein kinase is an antisense construct, and in another aspect this factor that binds and inactivates EGF-R protein kinase is an antibody or a small molecule inhibitor. In a particularly preferred embodiment, the factor is hydroxyurea, genistein, tyrphostin 1, tyrphostin 23, tyrphostin 63,
It may be selected from the group consisting of tyrphostin 25, tyrphostin 46 and tyrphostin 47.

[0016] Another aspect of the invention provides a method of providing a therapeutic polypeptide to a cell, comprising a polynucleotide encoding the polypeptide and a promoter active in a eukaryotic cell, wherein the promoter is Providing an AAV vector comprising an expression construct comprising the polynucleotide (operably linked to a promoter); contacting the cell with the vector under conditions that allow for uptake of the vector by the cell; Fibroblast growth factor receptor (FGFR) on surface
(Where an increase in FGFR results in increased uptake of the vector by the cell). In a preferred embodiment, the FGFR
The polypeptide is selected from the group consisting of FGFR1, FGFR2, FGFR3 or FGFR4. In certain embodiments, the therapeutic polypeptide is a hormone, tumor suppressor, inhibitor of apoptosis, toxin, lymphokine, growth factor, enzyme, DNA binding protein or single chain antibody. In certain preferred embodiments, the cells are located within a mammal. In certain embodiments, the cells are cancer cells. More specifically, the cells are derived from lung, breast breast, melanoma, colon, kidney, testis, ovary, lung, prostate, hepatic, reproductive cancer, epithelium, prostate,
Head and neck, pancreatic cancer, glioblastoma, astrocytoma, oligodendroglioma, ependymoma,
Selected from the group consisting of neurofibrosarcoma, meningia, liver, spleen, lymph nodes, small intestine, blood cells, colon, stomach, thyroid, endometrium, prostate, skin, esophagus, bone marrow and blood You.

Thus, in a broad sense, the present invention provides a method for treating a disease in a subject, comprising a therapeutic polynucleotide and a promoter active in a eukaryotic cell, wherein the therapeutic Providing an AAV vector comprising an expression cassette comprising a polynucleotide operably linked to a promoter; contacting the vector with a host cell under conditions that allow for uptake of the vector by the host cell; and Increasing the amount of FGFR on the surface of the cell;
(Where an increase in FGFR on the cell surface increases the ability of the cells to take up AAV). Thus, the AAV may provide a therapeutic polynucleotide to the subject's cells, wherein the polynucleotide is transcribed and has an effect in treating the disease. The disease can be any disease that can be treated by adaptation of the polynucleotide (eg, cystic fibrosis, cancer, hyperproliferative disorders, etc.).

[0018] Also provided herein is an adeno-associated virus expression construct comprising: a first polynucleotide encoding a selected gene and a first promoter functional in a eukaryotic cell, wherein: This polynucleotide is under the transcriptional control of a first promoter); and a second polynucleotide encoding FGFR. In a preferred embodiment, the FGFR polypeptide is FGFR1,
It is selected from the group consisting of FGFR2, FGFR3, or FGFR4. In certain embodiments, the expression construct may further comprise a third polynucleotide encoding an HSPG polypeptide. In a preferred embodiment, the FGFR encoding the polynucleotide is under the control of a first promoter. In other embodiments, the first and second polynucleotides are separated by an IRES. In certain embodiments, the second polynucleotide is under the control of a second promoter operable in eukaryotic cells. In particularly preferred embodiments, the selected gene is a tumor suppressor, cytokine, receptor,
It encodes a protein selected from the group consisting of an apoptosis inducer and a differentiation factor. In embodiments where the gene encodes a tumor suppressor, the tumor suppressor is p53, p16, p21, MMAC1, p73, zac1.
, C-CAM, BRCAI, and Rb. In embodiments where the gene encodes an inducer of apoptosis, the inducer of apoptosis may be selected from the group consisting of Harakiri, Ad E1B and ICE-CED3 protease. In embodiments where the gene encodes a cytokine, the cytokine is IL-2, IL-2, IL-3, IL-4, IL-4
-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, I
It is selected from the group consisting of L-12, IL-13, IL-14, IL-15, TNF, GMCSF, β-interferon, and γ-interferon. In embodiments where the gene encodes a receptor other than FGFR and HSPG,
The receptor may be selected from the group consisting of CFTR, EGFR, VEGFR, IL-2 receptor and estrogen receptor.

The present invention further provides a promoter and a first polynucleotide encoding a selected polypeptide that is functional in a eukaryotic cell, wherein the first polynucleotide is under the transcriptional control of the promoter. A first adeno-associated virus expression construct comprising; a second polynucleotide encoding FGFR; and a pharmaceutically acceptable buffer, solvent or diluent. In certain embodiments, the composition may further comprise a second expression construct comprising a third polynucleotide encoding an HSPG polypeptide, wherein the third polynucleotide comprises a third promoter. Operatively connected to

[0020] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description, so that the detailed description and specific examples illustrate preferred embodiments of the present invention. It should be understood that this is provided only by way of example.

Description of Illustrative Embodiments The use of viral vectors in various gene transfer attempts is now widely accepted. For example, retroviral vectors have been used for many years to transform cell lines in vitro to express exogenous polypeptides. More recently, along with advances in gene therapy, along with retroviruses, various other vectors, including adenovirus and herpes virus, and more recently, adeno-associated viruses have been utilized to transfer therapeutic genes to cells. I have.

While retroviral and adenoviral vectors have been associated with a wide variety of pathological indicators, adeno-associated virus (AAV) vectors may be particularly desirable for a number of reasons. In a first example, the AAV is:
It is not associated with any known pathological indicators. In addition, AAV can infect non-dividing cells (Kotin et al., 1990; Kotin et al., 1991; Samul).
ski et al., 1991) and also have tumor suppressive properties (Berns and Giraud, 1996). Recombinant AAV vectors that lack both the coding sequence of wild-type AAV but retain stable chromosomal integration and expression characteristics of the recombinant gene upon transduction, both in vitro and in vivo, can be produced (Bert).
Lan et al., 1996; Kearn et al., 1996; Ponazhagan et al., 1
997a).

[0023] Despite the fact that AAV is clearly an attractive alternative to other viral vectors, the use of AAV as a delivery vector is limited. The efficiency of AAV infection of cells is low even though AAV has a large breeding area that transcends the interspecies barrier (Muzyczka, 1).
992). One factor proposed to account for the broad host range is that cell surface heparan sulfate proteoglycan (HSPG) may be a receptor for AAV (Summerford and Samulski, 1998). However, our recent studies have demonstrated significant donor changes in the ability of AAV vectors to transduce CD34 ⁺ hematopoietic progenitor cells from primitive human bone marrow (Po
nnazhagan et al., 1997). AAV was demonstrated not to bind to CD34 ⁺ cells from about 50% of normal volunteer donors. Nevertheless, the lack of virus binding to cells was insufficient to explain that AAV could not infect cells. For example, in our preliminary experiments, we noted that mouse NIH3T3 cells could bind this virus efficiently but could not be transduced by AAV. Therefore, we started looking for putative cell surface coreceptors for effective infection by AAV. The present invention relates to A
Elucidation of coreceptors for AV infection. Methods and compositions relating to this discovery are described in further detail herein below.

A. Invention The cell surface heparan sulfate proteoglycan (HSPG) has been identified as a putative receptor for AAV infection (Summerford and Sam).
ulski, 1998), but the transduction efficiency of AAV vectors appears to vary greatly in different cells and tissues in vitro and in vivo. However, summarizing their findings, Summerford and Samulski suggest that HSPG is simply the first adhesion receptor for AAV-2, and that AAV adhesion and infection can also be mediated by an as yet unidentified receptor. There is said. In addition, they suggested that their study could indicate that HSPG was necessary for AAV infection, but could not indicate whether HSPG was sufficient for infection. The present inventors have concluded that cells expressing only the HSPG receptor cannot be infected by AAV. We have inferred that HSPGs may be responsible for AAV binding to cells, but that AAV entry into cells is mediated by another receptor.

The present invention indicates that cell surface expression of HSPG alone is insufficient for AAV infection, and AAV also expresses human fibroblasts as coreceptors for successful virus entry into host cells. Indicates that it requires a factor receptor (FGFR). We found that cells expressing neither HSPG nor FGFR were AAV
Did not bind and as a result proved to be resistant to infection by AAV. These non-permissive cells contain cDNA encoding mouse HSPG and human FGFR.
Followed by stable transfection with AAV vector. Furthermore, AAV infection of permissive cells known to express both FGFR and epidermal growth factor receptor (EGFR) is abrogated by cell treatment with basic fibroblast growth factor (bFGF). Growth factors (EG
It is not suppressed by using F).

Briefly, we have identified two non-permissive human cells (M07e and Raji).
) Followed by the introduction of two genes (mouse HSPG and human FGFR1) followed by A
It could be demonstrated that it could be transduced with AV. HSPG using Raji cells
It was interesting to note that expression of either the gene or the FGFR gene was insufficient to allow AAV binding and entry. On the other hand, in M07e cells, introduction of the HSPG gene was sufficient to allow AAV to bind to and invade these cells. In a closer experiment, it was noted that M07e cells express the endogenous FGFR gene. Therefore, a successful AAV
Binding and subsequent entry requires co-expression of both HSPG and FGFR, and is about the same as FGF ligand (Rapraeger et al., 1991, Le.
doux et al., 1992, Roghani and Moscatelli, 1992.
Givol and Yayon, 1992, Kan et al., 1993). However, interestingly, as determined by bFGF binding, M07e cells and R
Despite the co-expression of similar levels of HSPG and FGFR1 in aji cells, AAV transduction efficiencies in the two cell types were significantly different. In addition, AAV binding to the two cell types is also approximately the same, but with virus D
The extent of NA entry correlated well with transduction efficiency in the two cell types. Therefore, it remains that other cellular factors may be required for high efficiency infection by AAV (Mah et al., 1998).

It is known that NIH3T3 cells express both endogenous HSPG and FGFR genes, and that AAV can indeed bind to the muHSPG-muFGFR complex. Since AAV could not enter these cells, it seems reasonable to suggest that the specificity of virus entry depends on huFGFR. Two additional sets of data support this content. First, muHSP
The G gene is functional in human cells, and secondly, huFGF inhibits AAV binding as well as entry into otherwise permissive human cells. On the other hand, EGF
It has no effect on AAV binding to, or entry into, 293 cells that express high numbers of EGFR (Mah et al., 1998). Thus, the lack of an effect of EGF on AAV-mediated transduction of 293 cells was due to the lack of EGF in these cells.
Not due to the absence of R. HuFGF, which can bind to muFGFR, is also NI
AAV binding to H3T3 cells can be blocked. Further studies performed with NIH3T3 cells stably transfected with the huFGFR1 expression plasmid are likely due to either suboptimal cell surface expression of human proteins or some form of steric hindrance to the mouse counterpart. Thus, although at a lower level, resulted in increased AAV transduction efficiency. Based on all available information, we have proposed a model for AAV infection, which is shown in FIG. In this model, co-expression of cell surface HSPG and FGFR1 is required for successful AAV binding followed by virus entry (FIG. 6A), both of which are blocked by bFGF (FIG. 6B).

In view of these findings, it is now possible to anticipate the improved use of AAV vectors in human gene therapy. For example, ensuring that cells that are about to undergo AAV-mediated gene therapy express FGFR and HSPG, will ensure efficient binding of AAV and uptake of AAV, thereby reducing the efficacy of the applied therapy. Increase effectiveness. Methods and compositions for achieving such improved gene therapy are described in further detail herein below.

B. Receptors for AAV Infection We have identified coreceptors that are responsible for effective AAV infection. Recently, HSPG has been identified as a putative receptor for AAV (Summe
rford and Samulski, 1998). However, effective viral infection of host cells by AAV is likely due to at least two distinct cell surface macromolecules (
Receptor and co-receptor), each of which requires at least two steps-
It is clear that this is achieved with adhesion and ingress. We have developed AAV
FGFR was identified as an essential coreceptor required for infection. These two receptors and their role in AAV infection are discussed in more detail herein below.

Recently, although HSPG was suggested to be a receptor for AAV infection, mouse NIH3T3, which is known to express HSPG, was infected by AAV as discussed above. could not. The present invention describes putative coreceptors for AAV infection. Our studies (described in the Examples) have shown that this co-receptor is FGFR (Rapra
Eger et al., 1991; Ledoux et al., 1992; Roghani and Mo.
scatelli, 1992; Givol and Yayon, 1992; Kan.
Et al., 1993). The present invention describes a model for AAV infection (FIG. 6).
In this model, co-expression of cell surface HSPG and FGFR1 is required for successful AAV binding, followed by virus entry (FIG. 6A), both of which are blocked by bFGF (FIG. 6B).

(A. FGF Receptor) Fibroblast growth factor (FGF) has a diverse range of medical
(Basilico and Moscatelli, 1992). FGF utilizes a receptor system to activate signaling pathways (Klagsbrunn and Baird, 19
91; Ornitz et al., 1992; Yayon et al., 1991; Raprage.
r et al., 1991). A major component of this system is the family of signaling FGF receptors (FGFR). FGFR is a representative polypeptide growth factor receptor. These receptors usually have three major identifiable (iden
tiable) region. First is the extracellular region, which contains the domain that binds the polypeptide growth factor (ie, the ligand binding domain). The second region is a transmembrane region and the third is an intracellular region. Many of these receptors contain a tyrosine kinase domain in this intracellular region.
In accordance with the present invention, it is contemplated that FGFR family members may act as coreceptors for AAV infection.

[0032] The FGFR contains an extracellular ligand binding domain and an intracellular tyrosine kinase domain (Basilico and Moscatelli, 1992). The second component of this receptor system is heparin sulfate (HS) proteoglycans or related heparin-like molecules (which bind FGF to FGFR and
(Ornitz et al., 1992; Yay).
on et al., 1991). The mechanism by which heparin / HS activates FGF is unknown, but heparin, FGF and FGFR can form a trimolecular complex (Ornit
z et al., 1992). Heparin / HS may be able to interact directly with FGFR, which binds it to FGF (Kan et al., 1993). In addition, heparin / HS is
Can facilitate oligomerization of two or more FGF molecules, which can be important for receptor dimerization and activation (Ornitz et al., 1992)
.

Heparin / HS is a heterogeneously sulfated glycosaminoglycan consisting of repeating disaccharide units of hexuronic acid and D-glucosamine. Minimal and highly sulfated octasaccharide from heparin (Ornitz et al.
992) Fragments or decasaccharides (Ishihara et al., 1993)
It was previously reported that fragments were required for FGF to bind to FGFR.

The fibroblast growth factor receptor (FGF-R) protein binds to a family of related growth factor ligands, the fibroblast growth factor (FGF) family. This family of growth factors is characterized by amino acid sequence homology, heparin binding avidity, ability to promote angiogenesis, and mitogenic activity on cells of epithelial, mesenchymal and neural origin.

The FGF family includes acidic FGF (aFGF) and basic FGF (b
FGF) (Gospodarowicz et al., 1986); int-2 gene product (Moore et al., 1986); hst gene product or Kaposi's sarcoma FGF (A
nderson et al., 1988; Taira et al., 1987); FGF-5 (Zha
n et al., 1988); and keratinocyte growth factor (Rubin et al., 1989).
And FGF-6 (I. Marics et al., 1989). The effects of these FGFs are mediated by binding to specific high affinity cell surface receptors of about 145 kDa and about 125 kDa (Neufeld and Go).
spodarowicz, 1986; U.S. Patent No. 5,733,893). U.S. Patent Nos. 5,707,632; 5,229,501 and 5,783.
No. 683, each of which is hereby incorporated by reference in detail, describes methods and compositions related to the identification and purification of various fibroblast growth factor (FGF) receptors.

FGFRs have been shown to be expressed in every organ and tissue tested (Givol and Yayon, 1992), but their expression in skeletal muscle and their expression in neuroblasts and glioblasts of the brain The relative abundance of expression of E. coli was particularly well correlated, demonstrating the high efficacy of AAV-mediated transduction in these tissues in vivo (Qing et al., 1998). There are at least four distinct but related members in the FGFR family--FG
FR1 (Genbank registration number P11362; U23445; U22324;
(Each of which is specifically incorporated herein by reference)), FGFR2 (Genb
an accession number P21802; L49241; L49240; L49239; L
4923 (each specifically incorporated herein by reference)), FGFR3 (
Genbank accession numbers P22607; AF055074; Q61851, each of which is hereby incorporated by reference in detail, and FGFR4 (Genb
ank accession numbers AF031695; Q03142; P22455, each of which is hereby incorporated by reference in detail)-these individual members are
It appears that they may be useful, alone or in combination with one another, in facilitating successful infection by AAV (Rapraeger et al., 1991, Ledoux).
Et al., 1992, Roghani and Moscatelli, 1992, Giv.
ol and Yayon, 1992, Kan et al., 1993, Lee et al., 1989).
.

The four genes encode four forms of FGFR1-4. This form is 2
One or three extracellular immunoglobulin (Ig) -like loops (IgI-IgIII)
And a common structure composed of one intracellular tyrosine kinase domain. For FGFR1-3, alternative splicing of exons encoding extracellular regions results in multiple receptor forms (Johnson et al., 1991, G.
ivol and Yayon, 1992). Genomic construction of the third Ig-like loop
This leads to three receptor variants. The two forms across the membrane result from alternative splicing of the two exons (IIIb and IIIc) encoding the second half of loop III, while exon IIIb
And the selective polyadenylation site preceding IIIc is FGFR1 (IIIa
) Is used to produce a soluble form of In humans and mice, FGF
The mRNA transcript of the IgIIIa splice variant of R1 encodes a protein potentially without a hydrophobic transmembrane domain, and thus, the receptor (S
R) (Werner et al., 1992). Those skilled in the art will recognize various F
Guillonne provides a comprehensive discussion of GFR isoforms
See au et al. (1998, which is hereby incorporated by reference in detail).

(B. HSPG Receptor) Heparin sulfate proteoglycan (HSPG) plays an important biological role in the cell-matrix adhesion process and is an essential regulator (or receptor) of growth factor action Demonstrate well. Proteoglycans are
Proteins that are classified by the post-translational attachment of a polysaccharide glycosaminoglycan (GAG) moiety, each composed of repeating disaccharide units (
For a review, see Jackson et al., 1991; Kjellen and Lind.
ahl, 1991). These can be found to be associated with both the extracellular matrix and the plasma membrane.

The four major, widely distributed, membrane-bound GAGs include heparin / HS and chondroitin sulfate A-chondroitin sulfate C. These unbranched sulfated GAGs are defined by the repeating disaccharide units containing their chains, by their specific sulfation sites, and by their susceptibility to bacterial enzymes known to cleave unique GAG linkages. (Linhardt et al., 1986). All have varying degrees of sulfation resulting in high negative charge densities. Proteoglycans can be modified by more than one type of GAG and have diverse functions, including roles in cell adhesion, differentiation and proliferation. In addition, cell surface proteoglycans are known to act as cellular receptors for some bacteria and several animal viruses, including: (Rostand and Esko)
Foot and mouth disease type O virus (Jackson et al., 1996), HSV.
Types 1 and 2 (Sheih et al., 1992; WuDunn and Spear, 1
989) and dengue virus (Chen et al., 1997).

[0040] Summerford and Samulski (1998) recently described HSPGs.
Has been shown to serve as the primary adhesion receptor for AAV type 2 (AAV-2). Furthermore, their results indicate that the presence of HS GAG on the cell surface indicates that AA
5 shows that V directly correlates with the efficiency with which cells can be infected. From some observations, S
amulski and Summerford hypothesized that AAV-2 could use cell surface proteoglycans as receptors. First, they use AAV-2
Binds to a cellufine sulfate column. Other viruses are known to interact with columns that bind to negatively charged surface molecules (eg, some members of the Herpesviridae family known to use HS proteoglycans as attachment receptors (Compton et al.). Mettenleiter et al., 1990.
Sheih et al., 1992; WuDunn and Spear, 1989)). Second, AAV can infect a wide variety of human, rodent and monkey cell lines.
This suggests that AAV uses ubiquitous cell surface molecules for infection (Muzyczka, 1992; Berns, 1996). One such family of ubiquitous receptors is the proteoglycan family (Die
rtrich and Casaro, 1977; Kjellen and Lind
ahl, 1991). Despite these correlations, Samulski and S
Ummerford (1998) concluded that HS proteoglycans are required for AAV infection, but their studies did not focus on whether HS proteoglycans are indeed sufficient. We have shown that HSPGs mediate AAV binding to the cell surface, but they cannot mediate AAV entry into cells. We show for the first time that efficient AAV-mediated gene transfer requires FGFR as a co-receptor.

C. Adeno-Associated Virus and Its Use in Gene Therapy Vectors based on adeno-associated virus 2 (AAV) are useful for retroviral and adenoviral vectors more commonly used in gene therapy in humans. It is attracting attention as an alternative. Although AAV utilizes the ubiquitously expressed cell surface heparan sulfate proteoglycan (HSPG) as a receptor, the transduction efficiency of AAV vectors varies widely in vitro and in vivo in different cells and tissues. The present invention concludes that cell surface expression of HSPG alone is insufficient for AAV infection, and that AAV also translocates human fibroblast growth factor receptor 1 (FGFR1) into successful virus entry into host cells. Required as a co-receptor. The identification of FGFR1 as a co-receptor for AAV provides new insights not only about its role in the life cycle of AAV, but also about the optimal use of AAV vectors in human gene therapy. This section provides a discussion of the use of AAV in gene therapy applications.

(A. Adeno-Associated Virus) AAV utilizes linear, single-stranded DNA of about 4700 base pairs. The inverted terminal repeat is flanked by its genome. There are two genes in the genome, giving rise to a number of distinct gene products. The first cap gene produces three different virion proteins (VP), termed VP-1, VP-2 and VP-3. The second rep gene encodes four non-structural proteins (NS). One or more of these rep gene products are responsible for transactivating AAV transcription. The sequence of AAV is provided (Srivastava et al., 1983).

The AAV-ITR also contains an additional domain (a stretch of 20 nucleotides not involved in HP formation) termed the D sequence (Berns and Bohe).
nzky, 1987; Berns and Giraud, 1996; Srivas.
tava et al., 1983), we hypothesize that one or more cellular proteins interact with the D sequence and prevent second strand viral DNA synthesis. Thus, the identification of such host proteins deserves research. Once elucidated,
It is possible to increase transcription and replication from adeno-associated virus (AAV) vectors. Other uses for such proteins will become apparent in the disclosure below.

The three promoters in AAV are named by their position in the genome in map units. From left to right, p5, p19 and p40 are present. Transcription results in six transcripts, two initiated at each of the three promoters and one of each pair spliced. Map units 42 to 46
Is the same for each transcript. The four nonstructural proteins are clearly derived from the longer transcript, and all three virion proteins all result from the smallest transcript.

AAV is not associated with any pathological condition in humans. Interestingly,
For efficient replication, AAV requires "help" functions from viruses such as herpes simplex viruses I and II, cytomegalovirus, pseudorabies virus and, of course, adenovirus. The best characterized of the herpes virus is the adenovirus, and many "early" functions for this virus have been shown to support AAV replication. Low level expression of the AAV rep protein is thought to retain the structural expression of AAV in chickens, and helper virus infection is thought to remove this block.

The terminal repeats of the AAV vector of the present invention can be obtained by restriction endonuclease digestion of a plasmid containing the AAV or modified AAV genome (eg, p201) (Samulski et al., 1987), or other methods known to those skilled in the art. (AAV
Including, but not limited to, chemical or enzymatic synthesis of terminal repeats based on the published sequences of One of skill in the art can determine, by well-known methods (eg, deletion analysis), the minimum sequence or portion of the AAV ITR required to enable function (ie, stable and site-specific integration). . One of skill in the art can also determine which minor modifications of the sequence can be tolerated while maintaining the ability of the terminal repeat to direct stable site-specific integration.

B. Adeno-Associated Virus-Mediated Gene Therapy AAV-based vectors have proven to be safe and effective vehicles for gene delivery in vitro, and these vectors are currently being developed. And has been tested in preclinical and clinical stages for a wide range of potential applications in gene therapy, both ex vivo and in vivo. However, the present inventors (Ponazhagan et al., 1997b; 1997c; 1997).
d; 1997d) and others (Carter and Flotte, 1996; C;
hatterjee et al., 1995; Ferrari et al., 1996; Fisher
Flotte et al., 1993; Goodman et al., 1994; Ka.
Plitt et al., 1994; 1996, Kessler et al., 1996; Koebe.
rl et al., 1997; Mizukami et al., 1996; Xiao et al., 1996) repeatedly observed a wide variation in AAV transduction efficiency in different cells and tissues in vitro and in vivo.

The identity of putative cell surface receptors remains elusive (Miz
(Ukami et al., 1996), it seems plausible to suggest that AAV transduction efficiency correlates with the number of putative cell surface receptors. However, our work has revealed that such a correlation is probably not present. 293 expressing a relatively minimal number of these putative receptors
The cells transfected most efficiently. This is a previously published report (
Ferrari et al., 1996; Fisher et al., 1996).

AAV-mediated efficient gene transfer and expression in the lung has already led to clinical trials for the treatment of cystic fibrosis (Carter and Flott)
e, 1996; Flotte et al. (1993). Similarly, treatment of muscular dystrophy by AAV-mediated gene delivery of the dystrophin gene to skeletal muscle, treatment of Parkinson's disease by delivery of tyrosine hydroxylase gene to the brain, IX to the liver
Prospects for treatment of hemophilia B by gene delivery and potential myocardial infarction by the vascular endothelial growth factor gene to the heart are promising. Because
AAV-mediated transgene expression in these organs has now been found to be very efficient (Fisher et al., 1996; Flotte).
Kaplitt et al., 1994; 1996; Koeberl et al., 1;
997; McCown et al., 1996; Ping et al., 1996; Xiao et al., 19
96). Since the present invention shows that high efficiency of recombinant AAV transduction in these organs or tissues requires the presence of the FGFR co-receptor along with the presence of HSPG, any AAV-mediated gene therapy approach It is improved by ensuring the presence of these receptors on target cells. If such receptors are not expressed endogenously, they can be engineered to target cells / target organs, thereby ensuring efficient binding and uptake of the AAV vector.

D. Additional Factors Involved in Efficient AAV-Mediated Gene Transfer Another aspect of the present invention relates to post-receptor post-receptor cell events.
AA by manipulating the entry cellular event
For increasing v-mediated transgene expression. More specifically, embodiments of the present invention include:
Demonstrates our early findings that dephosphorylation of ssD-BP is required to allow AAV-mediated transgene expression and AAV transduction efficiency.

[0051] Dephosphorylation of D-BP results in AAV delivered to target cells as single-stranded DNA molecules.
Promotes second strand synthesis of the genome. This suggests that manipulation of the phosphorylation state of this protein can be developed as one of the strategies to significantly improve the transduction efficiency of recombinant AAV vectors. A strong correlation between the phosphorylation status of D-BP and the degree of efficiency of transduction by AAV in mouse organs / tissues in vivo has also been demonstrated, indicating that this improves transduction efficiency. We show that the approach works, and also show that D-BP can be evolutionarily conserved.

The mechanism by which D-BP dephosphorylation promotes second-strand viral DNA synthesis remains unclear. One possibility that D-BP itself may possess DNA polymerase-like activity is currently being tested. Alternatively, dephosphorylation of D-BP can
It can activate cellular DNA polymerases required for NA synthesis or DNA repair pathways, thereby achieving second strand viral DNA synthesis. Our work with highly purified preparations of D-BP shows that this protein undergoes autophosphorylation, followed by autophosphorylation, the significance of which is unclear. However, this purified D-BP was determined to be a protein of approximately 53 kDa but different from the p53 tumor suppressor protein. This is because the monoclonal anti-p53 antibody could not immunoprecipitate D-BP.

The present invention is based on the finding that the high efficiency of recombinant AAV transduction in various organs
Such an approach is also useful in determining the transduction potential of untested tissues / organs (especially of human origin) with AAV vectors, as shown by the best contribution to the presence of the dephosphorylated form of It is. For example, based on the data shown in Table 4, it appears that the kidney may be an additional organ selection for AAV-mediated transduction. Because, the ratio of dephosphorylated D-BP / phosphoric D-BP in these tissues is approximately 4 (level consistent with the ratio found in 293 cells, a cell line derived from human embryonic kidney).

In another aspect, searches for additional specific compounds that mediate D-BP dephosphorylation are facilitated by the present invention. The elucidation of such compounds serves to increase the transduction efficiency of recombinant AAV vectors in a wide variety of tissues and organs, including primary hematopoietic stem / progenitor cells. This potentially leads to their successful use in gene therapy for certain hematological disorders such as sickle cell anemia and beta thalassemia (Goodman et al., 1994; Ponnash
agan et al., 1997d; Walsh et al., 1994; Zhou et al., 1996).
Examples of mediators of phosphorylation known to those skilled in the art include genistein, tyrphostin A48, tyrphostin 1, tyrphostin 23, tyrphostin 25, tyrphostin 46, tyrphostin 47, tyrphostin 51, tyrphostin 63, tyrphostin AG 1478, herbmycin A, LY 29
4002, wortmannin, staurosporine, tyrphostin AG 126, tyrphostin AG 1288, tyrphostin 1295, and tyrphostin 1296
Is mentioned. Inhibitors of the tyrphostin group are believed to be particularly useful in combination with the present invention.

Previously, we have found that treatment of cells with a specific inhibitor of epidermal growth factor receptor protein tyrosine kinase (EGF-RPTK), such as tyrphostin, leads to a significant increase in AAV transduction efficiency. And ssD-
It has further been demonstrated that phosphorylation of BP is mediated by EGF-RPTK (U.S. Ser. No. 09 / 145,379, which is hereby incorporated by reference in its entirety) .Epidermal growth factor (EGF) Treatment of cells with ssD-BP results in phosphorylation of ssD-BP, while treatment with tyrphostin results in dephosphorylation of ssD-BP and subsequently leads to increased transgene expression. Transduction efficiency is inversely correlated with expression of EGF-R in different cell types, and EGF-R
Stable transfection of cDNA results in phosphorylation of ssD-BP,
Leads to significant inhibition in AV-mediated transgene expression, which can be overcome by tyrphostin treatment.

E. Gene Transfer and Expression a. Regulatory Elements Both AAV vectors containing the transgene of interest and any FGFR or HSPG receptor (including constructs for the purpose of protein expression) In writing, it should be noted that a promoter is required to drive transcription of these genes. The nucleic acid encoding the gene product is under the transcriptional control of a promoter. A "promoter" is a DNA recognized by a synthetic or induced synthetic machinery of a cell that is necessary to initiate specific transcription of a gene.
Refers to an array. The phrase "under transcriptional control" or "operably linked" means that the promoter is in the correct position and orientation with respect to the nucleic acid for controlling RNA polymerase initiation and expression of the gene.

In certain embodiments, an expression vector is used to express a receptor polypeptide for use in combination with AAV-mediated gene therapy. For expression, the appropriate signals are provided in a vector, and the vector comprises various regulatory elements, such as both viral and mammalian sources, that drive expression of the gene of interest in host cells. (Enhancer / promoter of interest). Elements that are designed to optimize the stability and translatability of the messenger RNA in the host cell are also defined. Conditions are also provided for the use of multiple dominant drug selections to establish permanent stable cell clones expressing the product. This is a factor that links the expression of the drug selectable marker for expression of this polypeptide.

Throughout this application, the term “expression construct” is used to refer to any type of genetic construct that includes a nucleic acid encoding a gene product, wherein some or all of the nucleic acid coding sequence is
(Which can be transcribed). Transcripts can be translated into proteins, but need not be. In certain embodiments, expression includes both transcription of the gene and translation of the mRNA into a gene product. In other embodiments, the expression comprises only transcription of the nucleic acid encoding the gene of interest.

The term promoter, as used herein, refers to a group of transcription control modules that are clustered around the start site of RNA polymerase II. Many considerations of how promoters are organized can be attributed to some viral promoters (
(Including promoters for HSV thymidine kinase (tk) and the SV early transcription unit). These studies (reinforced by more recent studies) show that promoters have individual functional modules (each of about 7-20
bp DNA and comprises one or more recognition sites for transcriptional activator or repressor proteins).

[0060] At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking the TATA box (eg, the promoter for the mammalian terminal deoxynucleotidyl transferase gene, and the promoter for the SV40 late gene). Individual elements beyond the start site itself help fix the location of the start.

[0061] Additional promoter elements regulate the frequency of transcription initiation. Typically,
Although they are located in the region 30-110 bp upstream of the start site, many promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is frequently flexible, so that promoter function is preserved when the elements are inverted or moved relative to each other. In the tk promoter, the interval between promoter elements can be increased to an interval of up to 50 bp, after which activity begins to decrease. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

The particular promoter used to control the expression of the nucleic acid sequence of interest is not considered important, as long as it can direct the expression of the nucleic acid in the target cell. Thus, when human cells are targeted, the nucleic acid coding region is preferably located adjacent to and under the control of a promoter that can be expressed in human cells. Generally speaking, such promoters can include either human or viral promoters.

In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, SV40 early promoter, Rous sarcoma virus long terminal repeat, rat insulin promoter, and glyceraldehyde-3-phosphate dehydrogenase are the codes of interest. It can be used to obtain high levels of expression of the sequence. The use of other viral promoters or mammalian cell or bacteriophage promoters, which are well known in the art to achieve expression of the coding sequence of interest, will result in sufficient levels of expression for a given purpose. If so, they are considered as well.

[0064] The use of promoters with well-known properties can optimize the level and pattern of expression of the protein of interest after transfection or transformation. In addition, selection of a promoter that is regulated in response to a particular physiological signal may allow for inducible expression of the gene product. Tables 2 and 3 list some elements / promoter that can be used to regulate the expression of the gene of interest in the context of the present invention. This list is not intended to be exhaustive of all possible elements involved in promoting gene expression, but merely for illustration.

An enhancer is a genetic element that increases transcription from a promoter located at different locations on the same molecule of DNA. Enhancers are well organized like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcribed proteins.

A fundamental difference between enhancers and promoters is operability. The enhancer region must be able to stimulate transcription a small distance as a whole; this need not be the case for the promoter region or its components.
On the other hand, a promoter must have one or more elements that direct the initiation of RNA at a particular site and in a particular orientation, while enhancers lack these properties. Promoters and enhancers are often redundant and contiguous, and are often found to have very similar modular mechanisms.

The following is a list of viral promoters, cellular promoters / enhancers and inducible promoters / enhancers that can be used in combination with the nucleic acid encoding the gene of interest in the expression construct (Tables 1 and 2). Furthermore, any promoter / enhancer combination (Eukaryotic Prom)
otor Data Base EPDB) can also be used to drive gene expression. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters when the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional gene expression construct.

[0068]

[Table 1]

[0069]

[Table 2] Where a cDNA insert is used, it is typically desirable to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be critical to the successful practice of the present invention, and any sequence such as human growth hormone and the SV40 polyadenylation signal may be used. Terminators are also contemplated as elements of the expression cassette. These elements increase the message level,
It can then act to minimize readings from the cassette to other sequences.

(B. Transgene Constructs) Transgene expression is driven by a selected promoter. The choice of promoter will depend on the polypeptide to be expressed, the target tissue and the purpose of the expression. For example, if the protein is simply produced and purified in vitro, a high level of promoter is utilized. Where the protein is toxic to cells, it may be desirable to regulate the expression of the protein such that cell growth is maximized prior to polypeptide expression. Where processing or secretion of this protein depends on a particular step in the host cell cycle, it may be desirable to use a promoter that is regulated in an appropriate, cell cycle-dependent manner.

(C. Selectable Markers) In certain embodiments of the invention, the cells comprise a nucleic acid construct of the invention, and the cell comprises a marker in the expression construct, in vitro or in vivo,
Can be identified. Such a marker confers an identifiable change on the cell, allowing easy identification of the cell containing the expression construct. Usually, the inclusion of a drug selectable marker assists in the cloning and selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. is there. Alternatively, an enzyme such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) can be used. Immunological markers can also be used. The selection marker used will not be critical as long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selectable markers are well known to those skilled in the art.

D. Multigene Constructs and IRES In certain embodiments of the present invention, the use of internal ribosome binding site (IRES) elements is used to generate multigene messages (ie, polycistronic messages). used. IRES elements can bypass the ribosome scanning model of 5 'methylation cap-dependent translation and initiate translation at internal sites (Pelletier and Sonenberg, 198).
8). IRE elements (Pelletier and Sonenberg, 1988) from two members of the picornavirus family (polio and encephalomyocarditis) and IRES from mammalian messages
(Macejak and Sarnow, 1991). IRES
Elements can be linked to a heterologous open reading frame. Multiple open reading frames can be transcribed together, each separated by an IRES, creating a polycistronic message. The IRES element allows each open reading frame to access the ribosome for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message.

[0073] Any heterologous open reading frame can be linked to the IRES element. This includes genes for secreted proteins, multi-subunit proteins, those encoded by independent genes, intracellular or membrane-bound proteins, and selectable markers. In this manner, the expression of several proteins can be simultaneously engineered into cells using a single construct and a single selectable marker.

E. Delivery of Expression Vectors The present application proposes the use of AAV expression vectors for the delivery of genes to specific host cells or target cells. However, as described above, such host cells also have an efficient A.F.A. so that the transgene can be efficiently taken up and expressed.
Requires the presence of FGFR and HSPG receptors for AV infection. Thus, to increase the efficiency of AAV-mediated gene delivery,
It is desirable to stimulate, increase or induce host cell FGFR receptor activity and HSPG receptor activity. This can be achieved by delivering a gene encoding a receptor for the target cell. This delivery can be achieved using viral or non-viral delivery vectors.

In one embodiment, it may be useful to use a virus other than AAV to deliver the FGFR receptor expression construct and the HSPG receptor expression construct. Such viruses can include viruses that enter cells via receptor-mediated endocytosis, integrate into the host cell genome, and stably and efficiently express viral genes (Ridgew).
ay, 1988; Nicolas and Rubenstein, 1988; Ba.
ichwal and Sugden, 1986; Temin, 1986). These DNA viruses include papovavirus (Simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baic
hwal and Sugden, 1986), adenovirus (Ridgeway).
Baichwal and Sugden, 1986) and retroviruses (Coffin, 1990; Mann et al., 1983; Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1992).
83).

In another embodiment, non-viral transfer is contemplated. Several non-viral methods for the transfer of HSPG receptor expression constructs and / or FGFR receptor expression constructs into cultured mammalian cells are contemplated by the present invention. These include calcium phosphate precipitates (Graham and Van D
er Eb, 1973; Chen and Okayama, 1987; Rippe
Et al., 1990), DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984).
), Direct microinjection (Harland and Weintraub, 1985), D
NA-loaded liposomes (Nicolau and Sene, 1982; Fraley)
1979) and lipofectamine-DNA complexes, cell sonication (Fec
heimer et al., 1987), gene bombardment using high-speed microprojectiles (Yang et al., 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques can be successfully adapted for use in vivo or ex vivo.

[0077] Once the expression construct is delivered intracellularly, the nucleic acid encoding the gene of interest can be located and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration can be in a homologous position and orientation via homologous recombination (gene replacement) or it can be integrated into a random, non-specific location (gene augmentation). In a still further embodiment, the nucleic acid can be stably maintained within the cell as a separate episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to allow for maintenance and replication independent or synchronized with the host cell cycle. How the expression construct is delivered to the cell and where the nucleic acid remains in the cell depends on the type of expression construct used.

In yet another embodiment of the invention, the expression construct comprising the receptor gene may simply consist of naked recombinant DNA or a plasmid. Transfer of the construct may be by any of the methods described above, which render the cell membrane physically or chemically permeable.
Can be implemented. This is particularly applicable for transfer in vitro, but it may be applied to in vivo use as well. Dubensky et al., (198
4) Successfully injected polyomavirus DNA in the form of calcium phosphate precipitate into the liver and spleen of adult and newborn mice, demonstrating active virus replication and acute infection. Benvenisty and Nesif (198
6) also demonstrated that the transfected gene was expressed by direct intraperitoneal injection of the plasmid precipitated by calcium phosphate. It is expected that DNA encoding the gene of interest may also be transferred in a similar manner in vivo and express the gene product.

[0079] In yet another embodiment of the invention, for transferring a naked DNA expression construct into a cell, it may include a particle bombardment. This method relies on the ability to accelerate DNA-coated microprojectiles to high speeds which allow them to penetrate cell membranes and enter the cell without killing the cell (Klein 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate a current, which in turn generates the driving force (Yang et al., 1990). The microprojectiles used have been constructed of biologically inert materials, such as tungsten or gold beads.

Organs of choice include rat, mouse liver, skin, and muscle tissue bombarded in vivo (Yang et al., 1990;
Zelenin et al., 1991). This may require surgical exposure of the tissue or cells (ie, ex vivo treatment) to eliminate any tissue intervening between the gun and the target organ. Again, DNA encoding a particular gene can be delivered via this method and is further included in the present invention.

In a further embodiment of the present invention, the included expression construct may be trapped within a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. These form spontaneously when the phospholipids are suspended in excess aqueous solution. This lipid component undergoes a self-rearrangement before forming a closed structure and traps water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Lipofectamine-DNA complexes are also contemplated.

The delivery of nucleic acids mediated by liposomes and the expression of foreign DNA in vitro have been very successful. Wong et al. (1980) describe cultured chicken embryos,
The potential of liposome-mediated delivery and expression of foreign DNA in HeLa and hepatome cells was demonstrated. Nicolau et al. (1987) achieved successful liposome-mediated gene transfer in rats following intravenous injection.

In certain embodiments of the invention, the liposome is a hemagglutinating virus (HVJ
) And a complex. This has been shown to facilitate fusion with the cell membrane and to facilitate entry of the liposome-encapsulated DNA into cells (Kaneda et al., 1989). In other embodiments, liposomes can be complexed with or used with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In a still further embodiment, the liposome can be complexed with both HVJ and HMG-1;
Or can be used with them. In that such expression constructs have been used successfully in transfer and expression of nucleic acids in vitro and in vivo.
These are then applicable to the present invention. If a bacterial promoter is used in the DNA construct, it is also desirable to include a suitable bacterial polymerase in the liposome.

Another expression construct that can be used to deliver a nucleic acid encoding a particular gene into a cell is a receptor-mediated delivery vehicle. They utilize the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Due to the cell type-specific distribution of various receptors, their delivery can be highly selective (Wu and Wu,
1993).

A vehicle for targeting a receptor-mediated gene generally consists of two components: a cell receptor-specific ligand and a DNA binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoids (ASOR) (Wu and Wu, 1987) and transferrin (Wa
gner et al., 1990). Recently, synthetic neoglycoproteins that recognize the same receptors as ASOR have been used as gene delivery vehicles (Ferkol et al.,
1993; Perales et al., 1994) and epidermal growth factor (EGF) have also been used to deliver genes to squamous cell carcinoma cells (Myers,
EPO 0273085).

[0086] In other embodiments, the delivery vehicle can include a ligand and a liposome. For example, Nicolau et al. (1987) used lactosylceramide, galactose-terminated asialgangliosides incorporated into liposomes, and observed increased uptake of the insulin gene by hepatocytes. Thus, nucleic acids encoding particular genes can also be specifically delivered to cell types such as lung cells, epithelial cells or tumor cells by a number of receptor-ligand systems, with or without liposomes. It is possible to get. For example, epidermal growth factor (EG
F) can be used as a receptor for mediated delivery of nucleic acid encoding the gene in a number of tumor cells that show up-regulation of the EGF receptor. Mannose can be used to target mannose receptors or hepatocytes. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T cell leukemia) and MAA (melanoma) can be used as targeting moieties as well.

In certain embodiments, gene transfer may be more easily performed under ex vivo conditions. Ex vivo gene therapy refers to the isolation of cells from the animal, the delivery of the nucleic acid to the cells in vitro, and the subsequent return of the modified cells to the animal. This may involve surgically removing the tissue / organ from primary cultures of animals or cells and tissues.

F. Transmission of AAV Vectors and Transformation of Host Cells The following is an illustrative description of the transmission of the AAV vectors of the invention, of course, the conditions described are exemplary only, and It is possible for those skilled in the art to modify these propagation conditions according to the specific needs in light of the disclosure of the invention. Plasmid transfer can be accomplished with any standard gene transfer mechanism: calcium phosphate precipitation, lipofection, electroporation, microprojectile bombardment, or other suitable means. Following transfer, the host cells can be further infected with a helper virus, and the virions are isolated and the helper virus is inactivated (eg, heated at 56 ° C. for 1 hour). The helper-free pool of virions obtained is used to infect appropriate target cells. Mature virions can be further isolated by standard methods (eg, calcium chloride centrifugation) and inactivate any contaminating adenovirus.

The function of the vectors of the invention (ie, the ability to mediate the transfer and expression of a heterologous gene in hematopoietic stem or progenitor cells) can be assessed by monitoring the expression of the heterologous gene in transduced cells. Clearly, the assay for expression depends on the nature of the heterologous gene. Expression can be monitored by various methods, including immunological, histochemical or activity assays. For example, Northern analysis can be used to assess transcription using a suitable DNA probe or a suitable RNA probe. If antibodies against the polypeptide encoded by the heterologous gene are available, Western blot analysis, immunohistochemistry or other immunological techniques can be used to assess the production of the polypeptide. Suitable biochemical assays can also be used where the heterologous gene is an enzyme. For example,
If the heterologous gene encodes antibiotic resistance, determination of the resistance of the infected cell to the antibiotic can be used to assess the expression of the antibiotic resistance gene.

[0090] Site-specific integration can be assessed, for example, by Southern blot analysis. DNA
Is isolated from cells transduced with the vectors of the present invention, digested with various restriction enzymes, and analyzed by Southern blot using an AAV-specific probe. A single band of hybridization demonstrates site-specific integration. Other methods known to those skilled in the art (eg, polymerase chain reaction of chromosomal DNA (
PCR) analysis) can be used to assess stable integration.

G. Cell Culture and Cell Selection In one embodiment, the present invention is directed to transforming cells for the production of a mammalian cell culture for use in the various therapeutic aspects of the present invention. Consider the use of AAV vectors. In order for the cells to survive in vitro and in contact with the expression construct, it is important to ensure that the cells are maintained by the correct proportions of oxygen and carbon dioxide and nutrients, but are protected from microbial contamination is necessary. Cell culture techniques are well documented and disclosed herein by reference (Freshner, 1992).

[0092] The construct encoding the protein of interest can be transfected into a suitable host cell with the viral vector described above, followed by culturing the cell under suitable conditions. Genes for virtually any polypeptide can be used in this manner. An example of a useful mammalian cell line is a cell that expresses the appropriate receptor for the B19 virus. These include cells from bone marrow cells, peripheral blood cells and fetal liver cells.

[0093] Bone marrow cells are isolated and expanded for hematopoietic stem cells (HSC) (eg, by fluorescent chromogenic cell sorting as described in Srivastava et al., (1988)). HSCs have the ability to self-renew and initiate long-term hematopoiesis and to differentiate into various hematopoietic lineages in vitro. HSCs are transfected with the vectors of the invention or infected by changing the concentration of virions containing the subject hybrid vector, and then evaluated for expression of the heterologous gene. Assays for expression depend on the nature of the heterologous gene. Expression can be monitored by various methods, including immunological, histochemical or activity assays. For example, Northern analysis can be used to assess transcription using a suitable DNA probe or a suitable RNA probe. If antibodies to the polypeptide encoded by the heterologous gene are available, Western blot analysis, immunohistochemistry or other immunological techniques can be used to assess the production of the polypeptide. Suitable biochemical assays can also be used when the heterologous gene is an enzyme. For example, if the heterologous gene encodes antibiotic resistance, the determination of the resistance of the infected cell to the antibiotic can be used to assess the expression of the antibiotic resistance gene.

An important consideration is the appropriate modification required for a particular polypeptide. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the expressed protein.

Examples of useful mammalian host cell lines include Vero and HeLa cells and Chinese hamster ovary cell lines, W138 cells, BHK cells, CO
There are S-7 cells, 293 cells, HepG2 cells, NIH3T3 cells, RIN cells and MDCK cells. In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the manner desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for the function of the protein. Different host cells
It has characteristic and specific mechanisms for post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.

Many selection systems are used to include, but are not limited to: t
HSV thymidine kinase gene, hypoxanthine guanine phosphoribosyltransferase gene and adenine phosphoribosyltransferase gene in k cells, hgprt cells, or aprt cells, respectively. Antimetabolite resistance may also be used as a basis for selection for gpt conferring resistance to mycophenolic acid; neo conferring resistance to aminoglycoside G418; and dhfr conferring resistance to hygro conferring resistance to hygromycin. .

[0097] Animal cells can be grown in vitro in two ways: through the bulk of the culture, as non-anchorage-dependent cells growing in suspension, or on solid substrates for cell growth. As anchorage-dependent cells that require attachment (ie, a monolayer form of cell proliferation).

Non-anchorage-dependent or suspension cultures from continuous established cell lines are most widely used as a means for large-scale production of cells and cell products. However, suspension culture cells have limitations (eg, tumorigenicity and lower protein production than adherent T cells).

[0099] Large-scale suspension culture of mammalian cells in a stirred tank is a common method for the production of recombinant proteins. Two suspension culture reactor designs are widely used (stirred reactors and airlift reactors). The agitated design has been successfully used with a capacity of 8000 liters for the production of interferon. Cells are grown in stainless steel tanks having a height to diameter ratio of 1: 1 to 3: 1. Culture is usually performed using a bladed disc.
k) or marine propeller pattern
mixed with one or more stirrers based on the attenuator. Stirrer systems that provide less shear than blades have been described. Agitation can be driven either directly or indirectly by a magnetically coupled drive. The indirect drive reduces the risk of microbial contamination through the seal of the agitator shaft.

[0100] Airlift reactors, also initially described for microbial fermentation and later adapted for mammalian culture, rely on a gas stream for both mixing the culture and sending oxygen. The gas stream enters the riser section of the reactor and circulates. The gas is released at the culture surface and produces a bubble-free denser liquid in the downcomer section of the reactor to move downward. The main effectiveness of this design is that it is simple and there is no need for mechanical mixing. Typically, the height to diameter ratio is 10: 1. Airlift reactors scale up relatively easily, deliver a sufficient amount of gas, and produce relatively low shear forces.

The antibodies of the present invention are particularly useful for isolating antigens by immunoprecipitation. Immunoprecipitation involves the separation of a target antigen component from a complex mixture and is used to discriminate or isolate small amounts of proteins. For isolation of membrane proteins, cells must be solubilized in detergent micelles. Nonionic salts are preferred. This is because other drugs (eg, bile salts) precipitate at acidic pH or in the presence of divalent cations. Antibodies and the use of antibodies are described further below.

H. Transgenes For gene therapy using AAV vectors, virtually any transgene may be utilized in the vectors described herein. In a preferred embodiment, the heterologous gene encodes a biologically functional protein (ie, a polypeptide or protein that acts on the cellular machinery of the cell in which the biologically functional protein is expressed). For example, a biologically functional protein can be an essential protein for the normal growth of cells or for maintaining the health of a mammal. A biologically functional protein can also be present in a mammal by supplying a deficient protein, by providing an increased amount of a protein that is under-produced in a mammal, or in a mammal. It can be a protein that improves the health of a mammal, either by providing a protein that inhibits or prevents the resulting unwanted molecule. Biologically functional proteins may also be useful proteins for developing new gene therapies or for exploratory studies to study cellular mechanisms.

The expression of some normally secreted proteins can be engineered into cells. CDNAs encoding many useful human proteins are utilized. Examples include soluble CD-4, factor VIII, factor IX, von Willebrand factor, TP
A, urokinase, hirudin, interferon, TNF, interleukin,
Hematopoietic growth factors, antibodies, albumin, leptin, transferrin, and nerve growth factor.

The expression of non-secreted proteins can be engineered into cells. 2 of such proteins
Two general classes can be defined. First, once expressed in a cell,
It is a protein that binds and stays with cells at various targets. These targets include the plasma, nucleus, mitochondria, endoplasmic reticulum, Golgi, secretory granule membrane and plasma membrane. Non-secreted proteins are both soluble and membrane-bound. The second class of proteins are proteins that normally bind to cells, but where these proteins have been modified to be secreted by cells. Modifications include site-directed mutagenesis or truncated expression of the engineered protein resulting in secretion of the protein, as well as creation of novel fusion proteins that result in secretion of normal non-secreted proteins.

P53 is currently recognized as a tumor suppressor gene (Montenarh, 1
992). High levels of mutant p53 have been found in many cells transformed by chemical carcinogenesis, ultraviolet irradiation, and some viruses, including SV40. The p53 gene is a frequent target of mutational inactivation in a wide variety of human tumors and has already been proven to be the most frequently mutated gene in common human cancers (Mercer, 1992). . p53 is human NS
More than 50% of CLC (Hollstein et al., 1991) and are mutated in a wide range of other tumors.

The p53 gene encodes a 393 amino acid phosphoprotein that can form a complex with host proteins (eg, large T antigen and E1B). This protein is found in normal tissues and cells, but in generally lower concentrations as compared to transformed cells or tumor tissue. Interestingly, wild-type p53 appears to be important in regulating cell proliferation and cell division. Overexpression of wild-type p53 may, in some cases, be antiproliferative in human tumor cell lines.
Can be seen. Thus, p53 may act as a negative regulator of cell proliferation (Wei
nberg, 1991), and may directly suppress uncontrolled cell growth, or may directly or indirectly activate genes that suppress this growth. Thus, deletion or inactivation of wild-type p53 can contribute to transformation. However, some studies indicate that the presence of mutant p53 may be necessary for full expression of the transforming ability of this gene.

[0107] Wild-type p53 is recognized as an important growth regulator in many cell types. Missense mutants are common for the p53 gene and are known to occur at at least 30 different codons, allowing dominant alleles that cause a shift in cellular phenotype without diminishing homozygosity. Create often. In addition, many of these dominant negative alleles are considered to be tolerated in organisms and transmitted in the germline. The various mutant alleles are thought to range from minimal impairment to strong penetrance (dominant negative alleles) (Weinberg, 1991).

Casey and co-workers have reported that transfection of DNA encoding wild-type p53 into two human breast cancer cell lines restores growth-suppressive control in such cells (Casey et al., 1991). Similar effects have also been demonstrated in transfection of wild-type p53 into human lung cancer cell lines,
It was not demonstrated in the mutant (Takahashi et al., 1992). p53
Are considered dominant over the mutant gene and are selected for growth when transfected into cells with the mutant gene. Normal expression of transfected p53 is not deleterious to normal cells with endogenous wild-type p53. Thus, such constructs can be utilized by normal cells without side effects. Thus, it is proposed that treatment of p53-related cancers with a wild-type p53 expression construct will reduce the number of malignant cells or the rate of growth of malignant cells. Furthermore, recent studies suggest that some p53 wild-type tumors are also susceptible to the effects of exogenous p53 expression.

The major transitions in the eukaryotic cell cycle are triggered by cyclin-dependent kinases, ie CDKs. One CDK, cyclin-dependent kinase 4 (CDK4
) Regulates progression throughout the G ₁ phase. The activity of this enzyme in late G ₁ can phosphorylate Rb. The activity of CDK4 is biochemically characterized as an activation subunit, a D-type cyclin and a repression subunit (eg, a protein that specifically binds and inhibits CDK4 and thus can regulate phosphorylation of Rb). is controlled by p16 ^INK4) has (Serrano et al., 1993; Serra
no et al., 1995). Since the p16 ^INK4 protein is a CDK4 inhibitor (Serrano, 1993), deletion of this gene can increase the activity of CDK4, resulting in hyperphosphorylation of the Rb protein. p16 is also known to regulate the function of CDK6.

P16 ^INK4 belongs to a newly described class of CDK inhibitory proteins, which also includes p16 ^B , p21 ^WAF1 , ^CIP1 , ^SDI1 , and p27 ^KIP1 . The p16 ^INK4 gene is located at 9p21, a chromosomal region frequently deleted in many tumor types. p
^Homozygous deletions and mutations of the ^16INK4 gene are frequent in human tumor cell lines. This evidence suggests that the p16 ^INK4 gene is a tumor suppressor gene.
However, this interpretation has been challenged by observation, and the frequency of changes in the p16 ^INK4 gene is ^lower in primary uncultured tumors than in cultured cell lines (Caldas et al., 1994; Cheng et al., 1994; Hussu
ssian et al., 1994; Kamb et al., 1994a; Kamb et al., 1994b;
Mori et al., 1994; Okamoto et al., 1994; Nobori et al., 199.
5; Orlow et al., 1994; Arap et al., 1995). Restoration of wild-type p16 ^INK4 function by transfection with a plasmid expression vector reduced colony formation by some human cancer cell lines (Okamoto, 1994;
Arap, 1995).

C-CAM is expressed on virtually all epithelial cells (Odin and Oblink, 1987). C-CAM (having an estimated molecular weight of 105 kD) was originally isolated from the plasma membrane of rat hepatocytes by reaction with specific antibodies that neutralize cell aggregation (Oblink, 1991). Recent studies indicate that structurally C-CAM belongs to the immunoglobulin (Ig) superfamily and its sequence is highly homologous to carcinoembryonic antigen (CEA) (Lin and Guidotti, 1989). Using the baculovirus expression system, Ch
eung et al. (1993a; 1993b and 1993c) describe the first of C-CAM.
Is important for cell adhesion activity.

Cell adhesion molecules or CAMs are known to participate in a complex network of molecular interactions that regulate organ development and cell differentiation (Edelman, 19).
85). Recent data suggests that abnormal expression of CAM indicates that some neoplasms (eg,
Is preferentially expressed in epithelial cells and is involved in the development of neoplasms of some species,
(Edel-reduced expression of cadherin) may be involved in tumorigenesis (Edel
man and Crossin, 1991; Fixen et al., 1991; Buss.
emakers et al., 1992; Matsura et al., 1992; Umbas et al., 1
992). Also, Giancotti and Ruoslahti (1990), an increase of alpha ₅ beta ₁ integrin expression by gene transfer, demonstrated that it is possible to reduce tumor formation in Chinese hamster ovary cells in vivo. C-CAM
Has now been shown to inhibit tumor growth in vitro and in vivo.

Other tumor suppressors that can be used in accordance with the present invention include: RB, APC, DCC, NF-1, NF-2, WT-1, MEN-I, ME
N-II, zac1, p73, BRCA1, VHL, FCC, MMAC1, MC
C, p16, p21, p57, C-CAM, p27 and BRCA2. I inducers of apoptosis (eg, Bax protease, Bak protease, Bcl
-X _s protease, Bik protease, Bid protease, Haraki
ri protease, Ad E1B protease, Bad protease and IC
E-CED3 protease) may likewise find use according to the invention.

The various enzyme genes are enzymes of interest according to the present invention. Such enzymes include cytosine deaminase, hypoxanthine-guanine phosphoribosyltransferase, galactose-1-phosphate uridyltransferase, phenylalanine hydroxylase, glucocerebrosidase, sphingomyelinase, α-L-iduronidase, glucose-6. -Phosphate dehydrogenase, HS
V thymidine kinase and human thymidine kinase.

[0115] Hormones are another group of genes that can be used in the vectors described herein. Includes growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone,
Leptin, adrenocorticotropin (ACTH), angiotensin I and II, β-endorphin, β-melanocyte stimulating hormone (
β-MSH), cholecystokinin, endothelin I, galanin, gastric inhibitory polypeptide (GIP), glucagon, insulin, lipotropin, neurophysin, somatostatin, calcitonin, calcitonin gene-related peptide (CGRP)
), Β-calcitonin gene-related peptide, malignant factor of hypercalcemia (1-
40), parathyroid hormone-related protein (107-139) (PTH-rP)
, Parathyroid hormone-related protein (107-111) (PTH-rP), glucagon-like peptide (GLP-1), pancreatastin, pancreatic peptide, peptide YY, PHM, secretin, vasoactive intestinal peptide (VIP), oxytocin ,
Vasopressin (AVP), vasotocin, enkephalinamide, metorphinamide, α-melanocyte stimulating hormone (α-M
SH), atrial natriuretic factor (5-28) (ANF), amylin, amyloid P component (SAP-1), corticotropin releasing hormone (CRH), (CR
H), growth hormone releasing factor (GHRH), luteinizing hormone releasing hormone (LH)
RH), neuropeptide Y, substance K (neurokinin A), substance P and thyroid stimulating hormone releasing hormone (TRH). CDNAs encoding many therapeutically effective human proteins are available. Other proteins include protein processing enzymes (eg, PC2 and PC3), and PAMs, transcription factors (eg, IPF1), and metabolic enzymes (eg, adenosine deaminase, phenylalanine hydroxylase, glucocerebrosidase).

[0116] Other classes of genes intended to be inserted into the vectors of the present invention include interleukins and cytokines. Interleukin 1 (IL-1), I
L-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL
-9, IL-10, IL-11, IL-12, GM-CSF and G-CSF.

Examples of diseases for which the viral vectors of the invention are useful include adenosine deaminase deficiency, human clotting factor IX deficiency in hemophilia B, and cystic fibrosis (these are cystic fibrosis transmembrane Involved in the replacement of a receptor gene). The vectors embodied in the present invention may also be used in the treatment of hyperproliferative disorders (eg, chronic rheumatoid arthritis or restenosis) due to the transfer of genes encoding angiogenesis inhibitors or cell cycle inhibitors. Transfer of a prodrug activator, such as the HSV-TK gene, can also be used in the treatment of hyperproliferative disorders, including cancer.

[0118] Other therapeutic genes can include genes encoding antigens, such as viral, bacterial, fungal or parasite antigens. Viruses include picornavirus, coronavirus, togavirus, flavivirus (flavivirus)
rviru), rhabdovirus, paramyxovirus, orthomyxovirus,
Bunyaviruses, allenviruses, reoviruses, retroviruses, papovaviruses, parvoviruses, herpes viruses, poxviruses, hepadnaviruses and spongy viruses. Preferred viral targets include influenza, herpes simplex viruses 1 and 2, measles, smallpox, polio or HIV. Pathogens include trypanosomes, tapeworms, roundworms, and helminths. Also, tumor markers such as fetal or prostate specific antigens can be targeted in this manner. Preferred examples include the HIV envelope proteins (HIV env Proteins) surface antigen and the hepatitis B surface antigen. Administration of the vector according to the present invention for vaccination purposes requires that the vector-associated antigen be sufficiently non-immune to allow long-term expression of the transgene for which a strong immune response is desired. Is done. Preferably, individual vaccinations are only needed infrequently (eg, once every year or every two years) and provide long-term immune protection against infectious agents.

[0119] Cells engineered to produce such proteins can be used for either production of proteins in vitro, or for cell-based therapy in vivo. In vitro production entails complete purification of the expressed protein, either from a pellet of cells for the protein remaining bound to the cells, or from conditioned media from cells secreting the engineered protein. In vivo cell-based therapies are based on either the secretion of the engineered protein or the beneficial effects of cells expressing the non-secreted protein.

[0120] It is also contemplated that the mutated, truncated, or fusion protein be engineered in cells. Examples of each type of manipulation that result in secretion of the protein are shown (Ferber et al., 1991; Mains et al., 1995). A review of the use of such proteins to study regulated secretory pathways is also given (Burgess and Kelly, 1987; Chavez et al.,
1994).

(I. Method of Modifying Receptor Activity) As described above, the present invention provides a method for increasing the transduction efficiency of AAV infection, for example, in gene therapy. These methods take advantage of our findings as described in detail herein. It is the HS for effective AAV infection
In addition to cell surface expression of PG, expression of the FGFR receptor is required. Therefore, it is an object of the present invention to utilize the knowledge of the present invention. This utilization basically takes two forms. First, the susceptibility of a cell to infection with AAV can be determined by observing the expression of the FGFR receptor on the cell, alone, or together with the observation of HSPG expression (US Pat. 5,
707,632). Second, in determining that one or both of these receptors are absent from the target cell, the target cell is rendered sensitive by increasing the presence of one or both of these receptors on the cell surface. It is possible to give.

There are at least three different ways through which cell surface FGFR receptor expression can be increased. First, one can simply increase the amount of mature endogenous FGFR receptor contained in the cell in anticipation of the consequences of increased cell surface expression. This can include increased processing of transcription, translation or post-translational modifications. For example, by reducing turnover or receptor cycles, FG
It may also include increasing the stability of the FR. Although the regulation of FGFR has not been fully elucidated, many cell surface receptor molecules exhibit complexes, autoregulatory mechanisms,
Thereby, the binding and internalization of the receptor-ligand complex stimulates increased transcription and translation of the receptor. Thus, a fruitful approach may include the use of FGFR peptides or analogs that cause increased expression of the receptor after internalization. Classic pharmaceuticals may also be utilized, affecting FGFR levels.

Second, FG promotes expression of increased amounts of receptor in expression constructs
A cell having the FR gene can be provided. Various aspects of this embodiment are described in detail throughout the relevant portions of the specification. Briefly, one can provide a target cell with the appropriate vector encoding the FGFR gene of choice under the control of a promoter active in the target cell. A promoter can stimulate transcription of the FGFR gene, either constitutively or under some form of induction, resulting in expression of the receptor. Subsequent infection of cells with AAV particles results in increased cell surface FGF
Should be enhanced by R.

Third, the ability of existing FGFR receptors to bind to AAV can be directly increased, probably by cells treated with agents that bind to FGFR proteins or closely related molecules. Many receptors exhibit an effect called "allosteric", where the binding of a ligand to one part of the molecule affects the structure and possibly the function of another part. In this embodiment, the FGF peptides can serve as themselves or as a useful model for other molecules that bind to FGFR and improve the ability of the receptor to bind to AAV.

J. Methods of Treatment The vectors of the present invention are therapies that consist of administering and increasing, stimulating or providing other functions FGFR or HSPG receptor activity / function to the host cell, Useful for gene therapy. In certain embodiments, the vectors of the present invention can direct the cell-specific expression of a desired gene, and are thus useful for treating dyschromatosis. Such diseases include thalassemia, sickle cell anemia, diabetes and cancer. In the context herein, a heterologous gene may be the abnormally produced gene in a disease state, the normal counterpart of a gene that is poorly produced (eg, β for treatment of sickle cell anemia). -Globin, and [alpha] -globin, [beta] -globin or [gamma] -globin in the treatment of thalassemia). A heterologous gene may encode an antisense RNA as described above. For example, α
-Globin is produced in excess β-globin in β-thalassemia. Thus, β-thalassemia can be treated according to the present invention by gene therapy using a vector, wherein the heterologous gene encodes an antisense RNA. The antisense RNA is selected to bind to the target sequence of α-globin mRNA to prevent translation of α-globin, or α-globin DNA which binds to prevent translation of α-globin DNA. Are selected to bind to the target sequence. In treating cancer, the heterologous gene may be a gene associated with tumor suppression (eg, the retinoblastoma gene, a gene encoding p53, p16, p21 or tumor necrosis factor).

The use of the vectors of the invention for the treatment of a disease, in one embodiment, comprises
Transduction of hematopoietic stem or progenitor cells using the vectors of the present invention, in combination with the provision of active receptors that mediate efficient infection of host cells by AV vectors. A by Infecting HSC or Progenitor Cells Using AAV Vector
After preparation of a mature virion containing the AV vector, transduction is achieved. The transduced cells can be located in the patient or in the transduced ex vivo, and are introduced or reintroduced into the patient, for example, by intravenous transfusion (Rosenb).
erg, 1996).

In an ex vivo embodiment, HSC cells or progenitor cells are provided by obtaining bone marrow cells from the patient, and optionally enrich the population of bone marrow cells for HSCs. HSCs are grown in mature virions at about 37 ° C. at about 1-2
Transfected by time or transduced by standard methods of infection. Stable formation of the viral genome is achieved at about 37 ° C. for about 1 week to about 1 month of HS.
Achieved by incubation of C. Stable site-specific integration and erythroid cell-specific expression are assessed as described above. After the transduced cells have been introduced into the patient, the presence of the heterologous gene product can be determined by appropriate assays for the gene product in the patient (eg, if expression is erythroid cell-specific, Is monitored or assessed (in peripheral red blood cells or bone marrow). As noted above, the particular assay will depend on the nature of the heterologous gene product and can be readily determined by one skilled in the art.

A. Combination Therapy The present invention provides a method for increasing the efficiency of AAV-mediated gene transcription by providing an expression construct encoding a therapeutic gene of interest in combination with FGFR and / or cell surface HSPG. It has been described. Effective gene transfer by AAV results from the presence of the FGFR co-receptor, which mediates the uptake of AAV once bound to HSPG. Thus, efficient transcription can be achieved with FGFR receptor or HSP
This can be achieved by providing an expression construct comprising a therapeutic transgene in combination with an expression construct comprising a G receptor, or both. Alternatively, host receptors may be stimulated, up-regulated, or otherwise provided with a stimulator of these receptors, combined with a transgene expression construct.
You can be encouraged to take advantage of AV.

Using the methods and compositions of the present invention to stimulate endogenous receptors,
Or, to provide for expression of such a receptor in a target cell, a "target" cell is generally contacted with a stimulator or expression construct, and the cell is contacted with a therapeutic transgene construct (gene therapy). . Such a composition comprises AAV
It can be provided in a combined effective amount to increase the efficiency of infection. This process can include simultaneously contacting the cell with gene therapy and a receptor encoding the construct. This can be accomplished by contacting the cell with a single composition containing both the therapeutic gene and the receptor gene, or by simultaneously contacting the cell with two separate compositions, where one composition Comprises a therapeutic expression construct and the other comprises a receptor expression construct.

[0130] In addition to ensuring efficient uptake of AAV, it may be beneficial to provide agents or agents suitable for use in therapy in combination with a therapeutic expression construct.

The present inventors have determined that the local or regional delivery of a vector construct to a patient in need of gene therapy in combination with receptor expression would allow therapeutically effective genes to alleviate clinical disease. We propose a very efficient way to deliver. Alternatively, systemic delivery of a therapeutic expression construct may be appropriate in certain circumstances (eg, where extensive metastasis occurs).

K. Formulations and Routes for Administration to Patients If clinical application is intended, the pharmaceutical composition (in a form suitable for the intended application)
Expression vectors, viral stocks, proteins, antibodies and drugs). Generally, this involves preparing a composition that is substantially free of pyrogens, as well as other impurities that can be harmful to humans or animals.

In general, it is desirable to use appropriate salts and buffers to stabilize the delivery vector and allow for uptake by the target cells. Buffers are also used when recombinant cells are introduced into a patient. The aqueous compositions of the present invention comprise an effective amount of the vector for the cells, which is dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such a composition is also called an inoculum. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or a human. . As used herein, "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. No. The use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional vehicle or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention is via any conventional route, so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical administration. Alternatively, administration can be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions are usually administered as the pharmaceutically acceptable compositions described above.

The active compounds can also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium that includes, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating (eg, lecithin), by the maintenance of the required particle size (in the case of dispersants), and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (eg, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.)
Can be caused by: In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents that delay absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in an appropriate solvent with various of the other ingredients enumerated above, if necessary.
Filter sterilized. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. . In the case of sterile powders for the preparation of a sterile injectable solution, the preferred methods of preparation are the vacuum drying and lyophilization techniques, which provide an active ingredient and any This results in a powder of the further desired component.

As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delays Agents and the like. The use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional vehicle or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

For oral administration, the polypeptides of the present invention can be incorporated with excipients and used in the form of non-ingestible gargles and dentifrices. Mouthwashes may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's solution). Alternatively, the active ingredients may be incorporated into a preservative, including borate, glycerin and potassium bicarbonate. The active ingredient can also be dispersed in a dentifrice, including: gels, pastes, powders and slurries. The active ingredient may be added to the paste dentifrice in a therapeutically effective amount, which paste dentifrice may include water, a binder, an abrasive, a flavoring, a foaming agent and a wetting agent.

The compositions of the present invention may be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed at the free amino groups of the protein), as well as inorganic (eg, hydrochloric or phosphoric) or organic acids (eg, acetic, oxalic, tartaric, mandel) Acid and the like). Salts formed with free carboxyl groups also include inorganic bases (eg, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or iron (III) hydroxide), and organic acids (eg, isopropylamine, trimethylamine). , Histidine, procaine, etc.).

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms (eg, injectable solutions, drug release capsules, etc.). For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. It should be. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage can be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of subcutaneous infusion liquid or injected at the site of injection (eg, , "Remingto
n's Pharmaceutical Sciences ", 15th Ed
ition, pages 1035-1038 and 1570-1580)
. Some variation in dosing will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in each case, determine the appropriate dose for the individual subject. In addition, for human administration, the preparations may contain FDA
As required by the Office of Biologics standard,
Standards for sterility, pyrogenicity, general safety and purity should be followed.

L. Examples The following examples are included to demonstrate preferred embodiments of the invention. The technology disclosed in the following examples presents the technology disclosed by the present inventor that works well in the practice of the present invention, and therefore can be regarded as constituting a preferred embodiment of the present invention. It should be understood by those skilled in the art.
However, one of ordinary skill in the art, in light of the present disclosure, will recognize that many changes can be made in the particular embodiments disclosed and depart from the concept, spirit and scope of the present invention. Similar or similar results should still be obtained without. More specifically, it is clear that certain agents, both chemically and physiologically related, can be substituted for the agents described herein, but achieve the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Example 1 Materials and Methods Cells, Plasmids and Viruses Human cervical cancer cell line HeLa, human adenovirus-transformed human embryonic kidney cell line 293, and mouse fibroblast NIH3T3 cells were American Ty
Obtained from pe Culture Collection (Rockville, MD). Human nasopharyngeal carcinoma cell line KB, human lymphoblastoma (lymphoblast)
oid) The cell line Raji and the human megakaryocytic leukemia cell line M07e were each isolated from Drs. Asok C.I. Antony, Zacharie A. Brahm
i, and HalE. Broxmeyer (Indiana Universal
site School of Medicine, Indianapolis
, IN). Monolayer cultures of HeLa, KB, 293 and NIH3T3,
And a suspension culture of M07e and Raji was supplemented with 10% fetal bovine serum (FB
S) and Iscove's modified Dulbecco 'supplemented with 1% antibiotics
s medium (IMDM). Recombinant mouse HSPG (Syndecan-
1) (Saunders et al., 1989) to Dr. Bradley B. Ol
win (University of Colorado, Boulder, C
O). SV40 of human fibroblast growth factor receptor 1 (FGFR1)
Recombinant plasmid pSV7d / hF containing promoter driven cDNA
GFR1 has been described previously (Johnson et al., 1990) and
r. Lewis T. Williams (University of Ca
lifonia, San Francisco, CA). A recombinant AAV plasmid, pCMVp-lacZ, containing the b-galactosidase (lacZ) gene driven by CMVp, has been described elsewhere (Ponnazhagan).
Et al., 1997, Ponazhagan et al., 1997). Wild-type (wt) and recombinant AAV vector (vCMVp-lacZ) stocks were generated and generated as previously described (Ponnazhagan et al., 1997, Qing et al., 1997,
Qing et al., 1998, Ponazhagan et al., 1997, Ponnazzh.
agan et al., 1997), CsCl equilibrium density gradient centrifugation.
Physical particle titers of wt and recombinant vector stocks were determined by quantitative DNA slot blot analysis (Kube and Srivas).
Tava, 1997). The ratio of physical particles to infectious particles (about 1000: 1), and the titer of contaminating wild-type AAV-like particles in the recombinant vector stock (about 0.01%
) Was determined as previously described (Wang et al., 1998).

AAV Binding Assay AAV binding studies were performed as previously described (Qing et al., 1998, Po.
nnazhagan et al., 1997). Briefly, 5 × 10 ⁴ cells were replaced with 1% BSA
Was washed twice with IMDM. 1 ml of IMDM containing 1% BSA is added to 1.5 ml
The cells were added to the cells for 90 minutes at 4 ° C. with either × 10 ⁸ particles of [ ³⁵ S] methionine-labeled wt AAV alone or with more than 100-fold unlabeled wt AAV particles. After incubation, cells were washed four times with IMDM containing 1% BSA and solubilized with 1 ml of 0.5N NaOH at room temperature for 30 minutes. The radioactivity of the lysate is measured and the specific binding is
Total activity-calculated as non-specific activity as previously described (Qing et al., 1998).

FGF Binding Assay FGF binding experiments were performed as previously described by Kan et al. (1993).
The following modifications were made. Briefly, 5 × 10 ⁴ cells were replaced with I containing 1% BSA.
Washed twice with MDM. 1 ml of IMDM containing 0.1% BSA was added to Amers
ham (Arlington Heights, IL), 0.5 ng /
ml of ¹²⁵ I-bFGF alone or a large excess of unlabeled bFGF (Sigma C
chemical Co. , St. Louis, MO) was added to all cells. Cells were incubated at room temperature for 90 minutes. After incubation, cells were loaded onto Whatman GF / C glass fiber filters and washed four times with IMDM containing 0.1% BSA to give free ¹²⁵ I-bFGF.
Was removed from cell-associated bFGF. Like monster radioactivity, Beckman Gam
Measured on a ma counter. Specific binding was calculated as total radioactivity-nonspecific radioactivity.

Stable Transfection with HSPG and / or FGFR1 Expression Plasmids Transfection of M07e and Raji cells with different expression plasmid DNAs was performed using the DMRIE-C reagent manufacturer (Gibco-BRL, Gra
nd Island, NY).
. Selectable marker genes (Hyg ^R and Neo ^R ) were inserted into the HSPG and FGFR1 cDNA expression plasmids, respectively, by standard cloning methods. G418, hygromycin, or both, were added 48 hours post-transfection at a final concentration of 400 mg / ml and 300 mg / ml, respectively, and individual drug-resistant cell clones were isolated 28 days after selection. Released.

Recombinant AAV Transduction Assay Approximately equal numbers of cells were washed once with IMDM and then infected with the various indicated particles / cells using the recombinant vCMVp-lacZ vector. After infection (
p. i. ) At 48 hours, cells were fixed and X-gal (5-bromo-4)
-Chloro-3-indolyl b-D-galactopyranoside) and count the number of blue cells, or cells were stained as previously described (Ponaz
Hagan et al., 1997, Qing et al., 1997, Qing et al., 1998, Po.
nnazhagan et al., 1997, Ponnazhagan et al., 1997), F.
Either analyzed by ACS. In some studies, following infection with the recombinant vCMVp-lacZ vector, cells were washed vigorously with PBS and low molecular weight DNA samples isolated from the same number of cells were isolated as previously described (Ponnazhagan Et al., 1997), analyzed on Southern blots using a lacZ DNA probe.

Cellular Tyrosine Kinase Inhibitors and Processing Conditions Specific inhibitors of cellular protein tyrosine kinases (genistein; S
igma Chemical Co. , St. Louis, MO), EGFR
Specific inhibitors of PTK (tyrphostin 1; Sigma), and FGFR
PTK (SU4989 and SU5402; Calbiochem, La J
olla, CA) using dimethyl sulfoxide (DMSO)
And store the stock solution at 4 ° C. before use in experiments.
Diluted with MDM. Cells were either mock-treated or treated separately at various concentrations of these compounds at 37 ° C. for 2 hours. After treatment, remove cells from PBS
, And were either mock infected or infected with the recombinant AAV vector as described above.

Example 2 Successful Infection of Cells by AAV Requires a Cell Surface Coreceptor By all previously published studies, cell types capable of binding AAV were infected by AAV. Has been established (Qing et al., 1998, Summer).
Ford and Samulski, 1998, Ponazhagan et al., 19
96). For example, although this transduction effect varies greatly, permissive human cells (eg, HeLa, KB, and 293) can bind to AAV but not permissive cells (
For example, M072e) cannot bind (FIG. 1A). But curiously,
We believe that mouse NIH3T3 cells, which can also bind AAV efficiently, are also transduced by a recombinant AAV vector (vCMVp-lacZ) containing the b-galactosidase gene driven by the cytomegalovirus immediate-early promoter. Note that it cannot be introduced (FIGS. 1B-1E). In various cell types, AA
Since the efficiency of V-mediated transgene expression depends on the phosphorylation status of cellular ssD-BP (Qing et al., 1998), we have previously shown that it enhances AAV-mediated transgene expression. Under the same conditions as those described (Mah et al., 1998).
Transduction studies were performed with eLa cells and NIH3T3 cells, with or without pretreatment with tyrphostin 1. These results are shown in FIGS. 1B to 1E. As can be seen, the low level of transgene expression in untreated HeLa cells (FIG. 1B) can be significantly increased after treatment with tyrphostin 1 (
In contrast to FIG. 1C), as previously observed (Mah et al., 1998), transgene expression could not be detected in NIH3T3 cells (FIG. 1D), and treatment with tyrphostin 1 resulted in these cells. Did not induce a significant response (FIG. 1E).
). The lack of this response in NHH3T3 cells indicates that treatment with tyrphostin 1
This was not due to failure to catalyze the dephosphorylation of D-BP. In both cell types, mainly as detected by electrophoretic mobility-shift assay (EMSA) (Qing et al., 1997, Qing et al., 1998).
This is because a dephosphorylated form of BP was present after treatment with tyrphostin 1. These results indicate that following the initial attachment of AAV to the cell surface via the HSPG receptor, the inventors postulate that the efficient entry of the virus requires the presence of a putative cell coreceptor. lead.

Example 3 Fibroblast Growth Factor Receptor 1 (FGFR1) Is Required for AAV Binding Considering the fact that HSPG is not sufficient for AAV entry into cells The present inventors have begun to investigate the coreceptors required for AAV infection. The inventor believed that AAV could utilize FGFR as a coreceptor for entry into host cells. To this end, M07e cells (Ponnazhagan et al., 1996), which are known to be non-permissive for AAV infection, are stably transformed either with the following expression plasmids, alone or in combination: Transfected: mouse (mu) HSPG; and human (hu) FGFR1. HS
Additional human lymphoblastoid cell lines (Raji) known to be negative for cell surface expression of both PG and FGFR (Lebakken and Rapraeger, 1996, Kiefer et al., 1990) were also identified as muH.
Stably transfected with SPG, huFGFR1, or both. Mock-transfected clones and three individual clones from M07e and Raji cells, respectively, were described previously (Qing et al., 1998;
(Mah et al., 1998; Kan et al., 1993) were separately analyzed for binding of ¹²⁵ I-bFGF and ³⁵ S-AAV. These results are shown in FIGS. 2A and 2B.
Shown in M07e cells known to lack HSPG expression (Barlett and Samulski, 1998) do not bind to bFGF, but do not bind to muHSPG.
It is interesting to note that it allows significant binding of bFGF after stable transfection with the cDNA. Low levels of bFGF binding also
Occurs in M07e cells stably transfected with uFGFR1 cDNA alone, and the degree of binding is significantly higher when M07e cells co-express HSPG and FGFR1 (FIG. 2A). These results suggest that M07e cells do express the endogenous FGFR gene. Mock-transfected Raji cells also did not bind to bFGF as expected, and only low levels of binding to bFGF were seen in Raji cells stably transfected with either the HSPG or FGFR1 expression plasmid alone. Is detected. H
In Raji cells co-expressing SPG + FGFR1, significant binding of bFGF occurred, further confirming the need for both HSPG and FGFR1 for ligand binding. Radiolabeled AAV to these two cell types
Is very similar to the pattern of bFGF binding,
Interesting (Figure 2B). Taken together, these data show that HSPG and FGFR
1 strongly suggests that both cell surface expression is required for successful binding of AAV to host cells.

Example 4 (Co-expression of HSPG and FGFR1 on the cell surface confers AAV infectivity to non-permissive cells) M07e cells stably transfected with HSPG, or FGFR1, or both It was then of interest to test whether Raji cells could be successfully transduced with the recombinant vCMVp-lacZ vector. Individual clonal isolates from both cell types were either mock-infected or infected with vCMVp-lacZ under the same conditions and subjected to fluorescent cytometric separation (F
ACS) was analyzed for transgene expression. Representative histograms of each of these clones are shown in FIGS. 3A-3H.

In mock-infected M07e cells, transgene expression rarely occurred, as expected, whereas only HSPG was expressed or HSP
It is clear that M07e cells co-expressing G + FGFR1 could be easily transduced by the recombinant AAV vector, whereas M07e cells expressing FGFR1 alone were not (FIGS. 3A-3D). M07e cells contain exogenous FGF
Because of the expression of the R gene, expression of endogenous HSPG is sufficient to render these cells permissive for AAV infection. On the other hand, Raji cells are exogenous HSPGs.
Only when the gene or the FGFR1 gene is expressed does not allow AAV-mediated transduction, but co-expression of HSPG + FGFR1 confers AAV infectivity on these cells despite their relatively low efficiency ( 3E-3H). R
The underlying mechanism of low efficiency transduction in aji cells was further investigated by examining the extent of viral DNA invasion as described in the methods.
Despite similar levels of virus binding (FIG. 2B), the degree of viral DNA entry into Raji cells co-expressing HSPG + FGFR1 is significantly lower than that in M07e cells for the two cell types. It is clear that this was well related to the efficiency of AAV transduction in E. coli. Additional individual clonal isolates from both cell types were also evaluated for AAV transduction efficiency. Table 3 shows the accumulated data. FGF
It is clear that clones of M07e cells and Raji cells co-expressing R1 + HSPG are transduced with an average transduction efficiency of about 70% and 10%, respectively. Taken together, these studies show that HSPG + FGFR
It is established that co-expression of 1 is required not only for binding to AAV, but also for entry of AAV into host cells.

Table 3. Effect of stable transfection of M07e and Raji cells with the various expression plasmid DNAs shown on AAV-mediated transgene expression.

[0154]

[Table 3] Example 5 (AAV-mediated transgene expression requires dephosphorylation of cellular ssD-BP) The present inventors have recently demonstrated that host cell proteins (single-chain D sequence binding proteins (ssD
(Referred to as D-BP)) was provided as evidence of a significant determinant of AAV transduction efficiency (Qing et al., 1997; Qing et al., 1988). The present inventors have also determined that ssD-BP is phosphorylated at tyrosine residues by the protein tyrosine kinase (PTK) activity of the cellular epidermal growth factor receptor (EGFR). Because treatment of cells with tyrphostin, a specific inhibitor of EGFR PTK, causes dephosphorylation of ssD-BP and is AAV-mediated,
It induces a significant increase in the expression of the transgene post-receptor (Mah et al., 1998). M07c cells and Ra
In order to evaluate whether the same mechanism is operating in ji cells, HSPG
These cell types stably co-transfected with the FGFR1 expression plasmid and the FGFR1 expression plasmid were isolated from human adenovirus 2 as previously described (Mah et al., 1998).
Mock infection or infection with the recombinant vCMVp-lacZ vector was performed either in the presence of co-infection with (Ad2) or pretreated with tyrphostin 1. Because ssD- in both cell types
BP exists predominantly in the phosphorylated state, and the efficiency of AAV-mediated transduction is
This is because it is about 2-3%. Transgene expression was analyzed by FACS as described above. These results are shown in FIGS. 4A to 4F. As expected, transgene expression rarely occurred in mock-infected M07e cells (FIG. 4A) and Raji cells (FIG. 4D), whereas each cell type co-expressing HSPG and FGFR1 had the presence of Ad2. It is interesting to note that either below or with pretreatment with tyrphostin 1 showed approximately the same efficiency of transgene expression. These studies were extended to include additional individual clonal isolates from both cell types. This cumulative data, shown in Table 4, further illustrates the post-receptor invasion (s
demonstrates that dephosphorylation of sD-BP is required to enable AAV-mediated transgene expression.

Table 4. Co-infection with human adenovirus 2 on AAV-mediated transgene expression in M07e and Raji cell clones stably co-transfected with muHSPG + huFGFR1 expression plasmid DNA
Or the relative effect of treatment with tyrphostin 1.

[0156]

[Table 4] Example 6 FGFR autophosphorylation is not required for AAV-mediated transduction Ligand binding to FGFR induces receptor dimerization, followed by receptor autophosphorylation of intracellular signaling molecules Induces an increase (Rapraeg
er et al., 1991; Ledoux et al., 1992; Roghani and Mosc
atelli, 1992, Givol and Yayon, 1992, Kan et al.,
1993), the next objective was to determine whether the tyrosine kinase (PTK) activity of the FGFR binding protein affected AAV-mediated transgene expression. Human 293 cells and HeLa cells (known to be permissive for AAV infection) are either mock treated or specific inhibitors of FGFR PTK activity (Strawn et al., 1996; Mohammadi et al., 1997).
), And then recombinant vCMV under the same conditions.
Infections were carried out with the p-lacZ vector and the extent of transgene expression was determined 48 hours after infection, as described above. The results of these studies indicate which FGFR
PTK inhibitors also showed no effect on AAV-mediated transgene expression. From these studies, the present inventors have found that FGFR PTK
We conclude that activity is not required for AAV-mediated transgene expression.

Example 7 FGF Treatment Disrupts AAV Binding to Non-Permissive and Permissive Cells and Suppresses Virus Entry into Permissive Cells As a co-receptor for AAV binding and / or AAV entry FGFR
To further substantiate the involvement of A.1, we have created a large excess of bF during AAV infection.
Non-permissive cells (eg, NIH3T3 cells) and permissive cells (eg, 2
(93 cells) was presumed to disrupt AAV binding and AAV infection, respectively. To this end, the following two sets of studies were performed. In the first set, binding studies were performed with radiolabeled AAV using NIH3T3 and 293 cells in the absence or presence of a large excess of FGF. Unlabeled wild type (
(wt) AAV or some additional controls including heparin (as a positive control) and EGF (as a negative control) were also included. The results of such a study are shown in FIGS. 6-A and 6-B. As expected (Summerford and Sanmlski, 1998), NIH3T3
It is clear that AAV binding to cells was inhibited by heparin, and bFGF also inhibited AAV binding to a significant extent, while EGF had no effect under the same conditions. Similarly, unlabeled wild-type AAV significantly inhibited binding of radiolabeled AAV to 293 cells, as expected. AAV binding to 293 cells was also reduced in the presence of bFGF. On the other hand, the same concentration of EGF
It had no significant effect on AV binding. In the second set of studies,
An equal number of 293 cells were transduced under the same conditions with the recombinant vCMVp-lacZ vector, either in the absence or presence of a large excess of bFGF or EGF. Transgene expression was determined 48 hours after transduction as previously described (Ponaz
Hagan et al., 1997) and evaluated by X-gal staining. The results of these studies are shown in FIGS. 5C-5H. AAV-mediated transduction of 293 cells (FIG. 5C)
Was determined to be inhibited by about 89% in the presence of bFGF (FIG. 5E), but only about 2% in the presence of EGF (FIG. 5G). Genistein (A
These assays (FIGS. 5D, 5F) were performed using 293 cells pre-treated with a specific inhibitor of cellular protein tyrosine kinase (Qing et al., 1997), which is known to increase AV transduction efficiency. And FIG. 5H) gave similar results (about 89% inhibition with bFGF; 0% inhibition with EGF).
This indicates that the lack of transgene expression in the presence of bFGF (FIG. 5F)
This shows that it was not caused by phosphorylation of ssD-BP in cells. Taken together, these results reliably demonstrate that the HSPG-FGFR1 interaction is important not only for successful binding, but also for AAV entry into cells.

Example 8 AAV Co-Receptor Activity of FGFR1, FGFR2, FGFR3 and FGFR4 The experiments of the previous example demonstrate that FGFR1 cDN under the control of the SV40 promoter.
A was performed. There are at least three additional but related members of the FGFR family, namely FGFR2, FGFR3 and FGFR4 (Hughes, 1997), which are mainly differentially expressed in human tissues. FGFR1 is abundantly expressed in brain neurons and cardiomyocytes, while FGFR2 expression is prominent in choroid plexus and glial cells. F
GFR3 expression is abundant in the intestine and growth plate, and FGFR4 is expressed in hepatocytes and adrenal glands (Gonza
lez et al., 1996; Ozawa et al., 1996; Olwin et al., 1989). Amino acid sequences within the intracellular kinase domain are highly variable between different FGFRs (14-79% homology) and are probably responsible for their different signaling and mitogenic capabilities (Wang et al., 1994). The present inventors have proposed Dr. Dan Donogue (University of Cali).
Fornia, San Diego) obtained expression plasmids for FGFR1, FGFR2, FGFR3 and FGFR4 cDNA, each under the control of the CMV promoter. Each plasmid was transferred to control Raji cells and HS
Transfected into Raji cells expressing PG, and clonal populations were analyzed for various types of FGFR for their potential co-receptor activity for AAV-mediated transduction. These results are shown in FIG. FGFR1 had the highest activity, followed by FGFR2 and FGFR3. In these experiments, we were unable to detect any co-receptor activity with FGFR4.

All compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.
Although the compositions and methods of the invention have been described with reference to preferred embodiments, the compositions and / or methods described herein may be changed without departing from the spirit, scope and scope of the invention. And may be applied in the steps of the method or in the order of the steps. More specifically, it is possible that certain agents, both chemically and physiologically related, could be substituted for the agents described herein while still achieving the same or similar results. it is obvious. Such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES The following references, to the extent that they provide details of the exemplary procedures set forth herein or which provide those details in addition to the other details set forth herein, are incorporated herein by reference. It is referred to in detail as.

[0161]

[Table 5] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood with reference to one or more of these drawings, in conjunction with the detailed description of the specific embodiments presented herein.

[Brief description of the drawings]

1A-1E: Analysis of binding of wt AAV to different cell types (FIG. 1A)
VCMVp-la in HeLa and NIH3T3 cells
Comparative analysis of transduction efficiency of cZ vector (FIGS. 1B-1E). The same number of human KB
Cells, HeLa cells, 293 cells, M07e cells, and mouse NIH3T3 cells were analyzed in triplicate in a binding assay using ³⁵ S-AAV as described in Methods. Approximately the same number of HeLa cells (FIGS. 1B and 1C) and NI
H3T3 cells (FIGS. 1D and 1E) are either mock treated (FIGS. 1B and 1D) or treated with 500 mM tyrphostin 1 for 2 hours (FIGS. 1C and 1E);
Either infection was performed with 2 × 10 ³ particles / cell of vCMVp-lacZ under the same conditions. Cells were fixed 48 hours post infection (pi), stained with X-gal, and photographed using a Nikon inverted light microscope. Magnification x 100.

FIGS. 2A and 2B: After either mock transfection or stable transfection with HSPG and / or FGFR1 expression plasmids.
¹²⁵ I-bFGF against M07e and Raji cells (FIG. 2A) and ³⁵
Comparative analysis of binding of S-AAV (FIG. 2B). Approximately the same number of cells were analyzed in triplicate as described in Example 1.

3A-3H: M07e cells (FIGS. 3A-3D) and Raji cells (FIG. 3)
E to FIG. 3H) Analysis of transgene expression. Using the indicated expression plasmids, infect the same number of mock or stably transfected cells with 10 ⁴ particles / cell of the vCMVp-lacZ vector under the same conditions, and As described, pACS by FACS for 48 hours. i. Was analyzed. For each sample, 1 × 10 ⁴ cells were analyzed. Table 3 provides the percentage of cells in the M1 region that express the transgene.

FIGS. 4A-4F: M07e cells (FIGS. 4A-4C) and Raji cells stably co-transfected with HSPG + FGFR1 expression plasmid in the presence of adenovirus co-infection or pretreatment with tyrphostin 1. Comparative analysis of transgene expression in (FIGS. 4D-4F). The same number of cells are recombined under the same conditions
The CMVp-lacZ vector was infected with 10 ⁴ particles / ml. 48 hours after infection, cells were analyzed by FACS as described in the legend to FIG. The data in FIGS. 4A and 4D show mock transduced cells. For each sample, 1 × 10 ⁴ cells were analyzed. Table 4 provides the percentage of cells in the M1 region that express the transgene.

5A-5H: A to non-permissive cells (FIG. 5A) and permissive cells (FIG. 5B).
Effect of bFGF and EGF on AV binding and on entry into permissive cells (FIGS. 5C-5H). ^{The 35} S-AAV binding assay was performed with a large excess of heparin (6 mg), bFGF (6 mg), EGF (6 mg), or unlabeled wtAAV.
The same number of NIH3T3 and 293 cells was performed essentially as described in the legend to FIG. 1, except that either (1 × 10 ¹⁰ particles) were included in the reaction mixture. The same number of 293 cells was also used, untreated (a), pretreated with 150 mM genistein (b), as well as in the presence of excess bFGF (c), 150 mM
Pretreatment with genistein and bFGF (d), also in the presence of excess EGF (
e) or with pretreatment (f) with 150 mM genistein and EGF,
Infected with 2 × 10 ³ particles / cell of the recombinant vCMVp-lacZ vector. Cells were fixed, stained, and photographed as described in the legend to FIG. Magnification x 40.

5A-5H: A to non-permissive cells (FIG. 5A) and permissive cells (FIG. 5B).
Effect of bFGF and EGF on AV binding and on entry into permissive cells (FIGS. 5C-5H). ^{The 35} S-AAV binding assay was performed with a large excess of heparin (6 mg), bFGF (6 mg), EGF (6 mg), or unlabeled wtAAV.
The same number of NIH3T3 and 293 cells was performed essentially as described in the legend to FIG. 1, except that either (1 × 10 ¹⁰ particles) were included in the reaction mixture. The same number of 293 cells was also used, untreated (a), pretreated with 150 mM genistein (b), as well as in the presence of excess bFGF (c), 150 mM
Pretreatment with genistein and bFGF (d), also in the presence of excess EGF (
e) or with pretreatment (f) with 150 mM genistein and EGF,
Infected with 2 × 10 ³ particles / cell of the recombinant vCMVp-lacZ vector. Cells were fixed, stained, and photographed as described in the legend to FIG. Magnification x 40.

5A-5H: A to non-permissive cells (FIG. 5A) and permissive cells (FIG. 5B).
Effect of bFGF and EGF on AV binding and on entry into permissive cells (FIGS. 5C-5H). ^{The 35} S-AAV binding assay was performed with a large excess of heparin (6 mg), bFGF (6 mg), EGF (6 mg), or unlabeled wtAAV.
The same number of NIH3T3 and 293 cells was performed essentially as described in the legend to FIG. 1, except that either (1 × 10 ¹⁰ particles) were included in the reaction mixture. The same number of 293 cells was also used, untreated (a), pretreated with 150 mM genistein (b), as well as in the presence of excess bFGF (c), 150 mM
Pretreatment with genistein and bFGF (d), also in the presence of excess EGF (
e) or with pretreatment (f) with 150 mM genistein and EGF,
Infected with 2 × 10 ³ particles / cell of the recombinant vCMVp-lacZ vector. Cells were fixed, stained, and photographed as described in the legend to FIG. Magnification x 40.

5A-5H: A to non-permissive cells (FIG. 5A) and permissive cells (FIG. 5B).
Effect of bFGF and EGF on AV binding and on entry into permissive cells (FIGS. 5C-5H). ^{The 35} S-AAV binding assay was performed with a large excess of heparin (6 mg), bFGF (6 mg), EGF (6 mg), or unlabeled wtAAV.
The same number of NIH3T3 and 293 cells was performed essentially as described in the legend to FIG. 1, except that either (1 × 10 ¹⁰ particles) were included in the reaction mixture. The same number of 293 cells was also used, untreated (a), pretreated with 150 mM genistein (b), as well as in the presence of excess bFGF (c), 150 mM
Pretreatment with genistein and bFGF (d), also in the presence of excess EGF (
e) or with pretreatment (f) with 150 mM genistein and EGF,
Infected with 2 × 10 ³ particles / cell of the recombinant vCMVp-lacZ vector. Cells were fixed, stained, and photographed as described in the legend to FIG. Magnification x 40.

5A-5H: A to non-permissive cells (FIG. 5A) and permissive cells (FIG. 5B).
Effect of bFGF and EGF on AV binding and on entry into permissive cells (FIGS. 5C-5H). ^{The 35} S-AAV binding assay was performed with a large excess of heparin (6 mg), bFGF (6 mg), EGF (6 mg), or unlabeled wtAAV.
The same number of NIH3T3 and 293 cells was performed essentially as described in the legend to FIG. 1, except that either (1 × 10 ¹⁰ particles) were included in the reaction mixture. The same number of 293 cells was also used, untreated (a), pretreated with 150 mM genistein (b), as well as in the presence of excess bFGF (c), 150 mM
Pretreatment with genistein and bFGF (d), also in the presence of excess EGF (
e) or with pretreatment (f) with 150 mM genistein and EGF,
Infected with 2 × 10 ³ particles / cell of the recombinant vCMVp-lacZ vector. Cells were fixed, stained, and photographed as described in the legend to FIG. Magnification x 40.

5A-5H: A to non-permissive cells (FIG. 5A) and permissive cells (FIG. 5B).
Effect of bFGF and EGF on AV binding and on entry into permissive cells (FIGS. 5C-5H). ^{The 35} S-AAV binding assay was performed with a large excess of heparin (6 mg), bFGF (6 mg), EGF (6 mg), or unlabeled wtAAV.
The same number of NIH3T3 and 293 cells was performed essentially as described in the legend to FIG. 1, except that either (1 × 10 ¹⁰ particles) were included in the reaction mixture. The same number of 293 cells was also used, untreated (a), pretreated with 150 mM genistein (b), as well as in the presence of excess bFGF (c), 150 mM
Pretreatment with genistein and bFGF (d), also in the presence of excess EGF (
e) or with pretreatment (f) with 150 mM genistein and EGF,
Infected with 2 × 10 ³ particles / cell of the recombinant vCMVp-lacZ vector. Cells were fixed, stained, and photographed as described in the legend to FIG. Magnification x 40.

5A-5H: A to non-permissive cells (FIG. 5A) and permissive cells (FIG. 5B).
Effect of bFGF and EGF on AV binding and on entry into permissive cells (FIGS. 5C-5H). ^{The 35} S-AAV binding assay was performed with a large excess of heparin (6 mg), bFGF (6 mg), EGF (6 mg), or unlabeled wtAAV.
The same number of NIH3T3 and 293 cells was performed essentially as described in the legend to FIG. 1, except that either (1 × 10 ¹⁰ particles) were included in the reaction mixture. The same number of 293 cells was also used, untreated (a), pretreated with 150 mM genistein (b), as well as in the presence of excess bFGF (c), 150 mM
Pretreatment with genistein and bFGF (d), also in the presence of excess EGF (
e) or with pretreatment (f) with 150 mM genistein and EGF,
Infected with 2 × 10 ³ particles / cell of the recombinant vCMVp-lacZ vector. Cells were fixed, stained, and photographed as described in the legend to FIG. Magnification x 40.

5A-5H: A to non-permissive cells (FIG. 5A) and permissive cells (FIG. 5B).
Effect of bFGF and EGF on AV binding and on entry into permissive cells (FIGS. 5C-5H). ^{The 35} S-AAV binding assay was performed with a large excess of heparin (6 mg), bFGF (6 mg), EGF (6 mg), or unlabeled wtAAV.
The same number of NIH3T3 and 293 cells was performed essentially as described in the legend to FIG. 1, except that either (1 × 10 ¹⁰ particles) were included in the reaction mixture. The same number of 293 cells was also used, untreated (a), pretreated with 150 mM genistein (b), as well as in the presence of excess bFGF (c), 150 mM
Pretreatment with genistein and bFGF (d), also in the presence of excess EGF (
e) or with pretreatment (f) with 150 mM genistein and EGF,
Infected with 2 × 10 ³ particles / cell of the recombinant vCMVp-lacZ vector. Cells were fixed, stained, and photographed as described in the legend to FIG. Magnification x 40.

FIGS. 6A and 6B: Models for the role of cell surface HSPGs and FGFR1 in mediating AAV binding and invading host cells. HSPG and FG
Co-expression of FR1 is required for successful AAV binding followed by virus entry into susceptible cells (FIG. 6A), both of which are disturbed by the ligand bFGF, which also inhibits HSPG-FGFR1 interaction. Required (FIG. 6B).

FIG. 7: AAV co-receptor activity of FGFR1, FGFR2, FGFR3, and FGFR4. These assays were performed as described in Example 1.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 1/16 A61P 1/18 1/18 7/00 7/00 11/00 11/00 13/08 13 / 08 13/12 13/12 15/00 15/00 17/00 17/00 19/00 19/00 35/00 35/00 A61K 35/76 // A61K 35/76 C12N 15/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW) , EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL , AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK , MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Chin, Kayun United States Indiana 46226, Indianapolis, Apartment D, Brendon Way Parkway 5641 (72) Inventor Mar, Kathryn United States Indiana 46254, A Indianapolis, Apartment Dee, Chipmunks Run 5779 (72) Inventor Hansen, Jonathan United States of America Indiana 46214, Indianapolis, Hunters Pass 7893 (72) Inventor Chou, Schanzen United States of America 94502, Alameda, Fundy Bay 125 (72) ) Inventor Dwaraki, Barabani, California 94502, Alameda, Old Alameda point 1177 F-term (reference) 4B024 AA01 AA20 BA63 CA01 DA03 EA02 FA02 FA20 GA11 GA18 HA17 4C084 AA13 MA02 NA14 ZA361 ZA591 ZA661 ZA751 ZA8 ZA8 ZA96 MA05 NA14 ZA36 ZA51 ZA59 ZA66 ZA75 ZA81 ZA89 ZA96 ZB26 4C087 AA02 BC83 MA02 NA14 ZA36 ZA51 ZA59 ZA66 ZA75 ZA81 ZA89 ZA96 ZB26

Claims (74)

[Claims] 1. The fibroblast growth factor receptor (FGFR) on the surface of a cell
A method of increasing AAV infection of said cells, comprising increasing the amount of AAV, wherein said increased FGFR increases AAV uptake by said cells. 2. The method of claim 1, further comprising contacting the cell with an AAV vector. 3. The method of claim 1, wherein the FG of the cell is
Increasing the amount of FR provides the cell with an expression construct comprising a polynucleotide encoding a FGFR polypeptide and a promoter active in eukaryotic cells, wherein the polynucleotide is operable with the promoter. A method comprising the steps of: 4. The cell surface heparan sulfate proteoglycan (HS) on the cell
2. The method of claim 1, further comprising increasing the amount of PG). 5. The method of claim 1, wherein said HS of said cells is used.
The step of increasing PG provides the cell with an expression construct comprising an HSPG polypeptide and a polynucleotide encoding a promoter active in eukaryotic cells, wherein the polynucleotide is operably linked to the promoter. The method comprising the steps of: 6. The method of claim 2, wherein said AAV is a vector comprising an expression cassette comprising a selected polynucleotide and a promoter active in eukaryotic cells, wherein said polyavonic acid comprises A method wherein a nucleotide is operably linked to the promoter. 7. The method of claim 6, wherein said selected polynucleotide encodes a polypeptide. 8. The method of claim 6, wherein said selected polynucleotide encodes an antisense construct. 9. The method of claim 6, wherein said selected polynucleotide encodes a ribozyme. 10. The method of claim 3, wherein said promoter is an inducible promoter. 11. The method according to claim 11, wherein the promoter is CMV IE promoter, SV4.
0 IE promoter, HSV tk promoter, β-actin promoter, human globin α promoter, human globin β promoter, human globin γ
Promoter, RSV promoter, B19p6 promoter, α-1 antitrypsin promoter, PGK promoter, tetracycline promoter,
4. The method according to claim 3, which is an MMTV promoter or an albumin promoter. 12. The method of claim 3, wherein said expression cassette further comprises a polyadenylation signal. 13. The method of claim 12, wherein said polyadenylation signal is an AAV polyadenylation signal, an SV40 polyadenylation signal or a BGH polyadenylation signal. 14. The method of claim 7, wherein the polypeptide is a hormone, a tumor suppressor, an inhibitor of apoptosis, a toxin, a lymphokine, a growth factor, an enzyme, a DNA binding protein or a single chain antibody. 15. The method according to claim 5, wherein said promoter is an inducible promoter. 16. The method according to claim 16, wherein the promoter is CMV IE promoter, SV4
0 IE promoter, HSV tk promoter, β-actin promoter, human globin α promoter, human globin β promoter, human globin γ
Promoter, RSV promoter, B19p6 promoter, α-1 antitrypsin promoter, PGK promoter, tetracycline promoter,
The method according to claim 5, which is an MMTV promoter or an albumin promoter. 17. The method of claim 5, wherein said expression cassette further comprises a polyadenylation signal. 18. The method of claim 17, wherein said polyadenylation signal is an AAV polyadenylation signal, an SV40 polyadenylation signal or a BGH polyadenylation signal. 19. The method of claim 5, wherein said expression construct is a viral vector. 20. The method of claim 19, wherein said viral vector is selected from the group consisting of a retrovirus, an adenovirus, a vaccinia virus, a herpes virus, and an adeno-associated virus. 21. The method of claim 6, wherein said promoter is an inducible promoter. 22. The promoter is a CMV IE promoter, SV4
0 IE promoter, HSV tk promoter, β-actin promoter, human globin α promoter, human globin β promoter, human globin γ
Promoter, RSV promoter, B19p6 promoter, α-1 antitrypsin promoter, PGK promoter, tetracycline promoter,
The method according to claim 6, which is an MMTV promoter or an albumin promoter. 23. The method of claim 6, wherein said expression cassette further comprises a polyadenylation signal. 24. The method of claim 17, wherein the polyadenylation signal is an AAV polyadenylation signal, an SV40 polyadenylation signal, or a BGH polyadenylation signal. 25. A method of expressing a polynucleotide selected from an adeno-associated virus (AAV) vector in a host cell, comprising the steps of: (i) selecting said polynucleotide and eukaryotic cell. Providing an AAV vector comprising an expression cassette comprising an active promoter, wherein the selected polynucleotide is operably linked to the promoter; (ii) allowing uptake of the vector by the host cell. Contacting said vector with said host cell under conditions that include: and (iii) increasing the amount of fibroblast growth factor (FGFR) on the surface of said cell, wherein said increasing is performed. A method wherein the altered FGFR increases AAV uptake by the cell. 26. The method of claim 25, wherein the FGF of the cell is
Increasing R provides the cell with an expression construct comprising an FGFR polypeptide and a polynucleotide encoding a promoter active in eukaryotic cells, wherein the polynucleotide is operable with the promoter. The method is linked to 27. The method of claim 25, further comprising increasing the amount of cell surface heparin sulfate proteoglycan (HSPG) in said cells. 28. The method of claim 25, wherein the HSP of the cell
G providing an expression construct comprising an HSPG polypeptide and a polynucleotide encoding a promoter active in eukaryotic cells to the cell, wherein the polynucleotide is operably linked to the promoter. how to. 29. A polynucleotide encoding said HSPG and said FPG
29. The polynucleotide encoding GFR is in the same expression construct.
The method described in. 30. A polynucleotide encoding said HSPG and said FPG
3. The polynucleotide encoding GFR is separated by an IRES.
9. The method according to 8. 31. A polynucleotide encoding said HSPG and said FPG
29. The method of claim 28, wherein the polynucleotides encoding GFR are under the control of separate promoters, each operable in a eukaryotic cell. 32. The method of claim 25, wherein said host cell is a eukaryotic cell. 33. The eukaryotic cell is a human red blood cell.
The method described in. 34. The method of claim 25, wherein said host cells are selected from the group consisting of bone marrow cells, peripheral blood cells, lung cells, gastrointestinal cells, endothelial cells and cardiomyocytes. 35. The method of claim 25, wherein said host cell is in an animal. 36. The method of claim 25, further comprising inhibiting a function of a D sequence binding protein (D-BP) in said host cell. 37. The method of claim 36, wherein said inhibiting comprises reducing the expression of D-BP in said host cell. 38. The method of claim 37, wherein reducing the expression of D-BP is accomplished by contacting the host cell with an antisense D-BP polynucleotide. 39. The method of claim 38, wherein said antisense D-BP polynucleotide targets a translation initiation site. 40. The method of claim 38, wherein said antisense D-BP polynucleotide targets a splice site. 41. The method according to claim 36, wherein the factor that reduces the expression of D-BP is an antibody or a small molecule inhibitor. 42. The method of claim 41, wherein said antibody is a single chain antibody. 43. The method 1 of claim 41, wherein said antibody is a monoclonal antibody. 44. The method of claim 36, wherein said inhibiting comprises reducing the D sequence binding activity of said D-BP in said host cell. 45. The method of claim 44, wherein reducing the binding activity is achieved by inhibiting tyrosine phosphorylation of D-BP. 46. The method of claim 45, wherein inhibiting phosphorylation is achieved by contacting the host cell with a D-BP peptide containing a tyrosine residue. 47. The method of claim 45, wherein inhibiting phosphorylation is accomplished by contacting the host cell with an agent that inhibits tyrosine kinase. 48. The method of claim 47, wherein said tyrosine kinase is an EGF-R tyrosine kinase. 49. The method of claim 48, wherein said factor is an inhibitor of EGF-R that reduces expression of EGF-R protein kinase. 50. The inhibitor of EGF-R protein kinase, wherein the inhibitor is EGF.
49. An agent that binds to and inactivates -R protein kinase.
The method described in. 51. The inhibitor of EGF-R protein kinase is DB
50. The method of claim 48, wherein the method inhibits the interaction of EGF-R with P. 52. The method of claim 50, wherein said factor that reduces EGF-R protein kinase expression is an antisense construct. 53. The method of claim 50, wherein said factor that binds and inactivates EGF-R protein kinase is an antibody or a small molecule inhibitor. 54. The method of claim 53, wherein said antibody is a single chain antibody. 55. The method of claim 53, wherein said antibody is a monoclonal antibody. 56. The method of claim 47, wherein said factor is selected from the group consisting of hydroxyurea, genistein, tyrphostin 1, tyrphostin 23, tyrphostin 63, tyrphostin 25, tyrphostin 46, and tyrphostin 47. 57. A method for providing a therapeutic polypeptide to a cell, comprising: (i) an AAV vector comprising an expression construct comprising said polypeptide and said polynucleotide encoding a promoter active in eukaryotic cells. To provide
Wherein said polynucleotide is operably linked to said promoter; (ii) contacting said vector with said cell under conditions allowing uptake of said vector by said cell; and (iii) Increasing the amount of fibroblast growth factor (FGFR) on the surface of the cell, wherein increasing the FGFR results in increased uptake of the vector by the cell. 58. The method of claim 57, wherein said therapeutic polypeptide is a hormone, a tumor suppressor, an inhibitor of apoptosis, a toxin, a lipokine, a growth factor, an enzyme, a DNA binding protein or a single chain antibody. 59. The method of claim 57, wherein said cells are located in a mammal. 60. The method of claim 59, wherein said cells are cancer cells. 61. The cancer cell may be lung, breast, melanoma, colon, kidney, testis, ovary, lung, prostate, liver, reproductive cancer, epithelium, prostate, head and neck, pancreatic cancer, glioblastoma, Astrocytoma, oligodendroglioma, ependymoma of the ventricle, neurofibrosarcoma, meningioma, liver, spleen, lymph node, small intestine, blood cells, colon, stomach, thyroid, endometrium, prostate, skin ,esophagus,
61. The method of claim 60, wherein the method is selected from the group consisting of bone marrow and blood. 62. An adeno-associated virus expression construct, comprising: (a) a first polynucleotide encoding a selected gene and a first promoter functional in a eukaryote, wherein An adeno-associated virus expression construct, comprising: a first polynucleotide, wherein the polynucleotide is under the transcriptional control of the first promoter; and (b) a second polynucleotide encoding FGFR. 63. The expression construct of claim 62, further comprising a third polynucleotide encoding an HSPG polypeptide. 64. The expression construct of claim 62, wherein said FGFR encoding polynucleotide is under the control of said first promoter. 65. The expression construct of claim 62, wherein said first polynucleotide and said second polynucleotide are separated by an IRES. 66. The expression construct of claim 62, wherein said second polynucleotide is under the control of a second promoter operable in eukaryotic cells. 67. The expression construct of claim 62, wherein said selected gene encodes a protein selected from the group consisting of a tumor suppressor, a cytokine, a receptor, an inducer of apoptosis, and a differentiation factor. 68. The tumor suppressor is p53, p16, p21, MM
68. The expression construct of claim 67, wherein the expression construct is selected from the group consisting of AC1, p73, zacl, C-CAM, BRCAI, and Rb. 69. The apoptosis inducer may be Harakiri,
Selected from the group consisting of Ad E1B and ICE-CED3 protease;
An expression construct according to claim 67. 70. The cytokine, wherein the cytokine is IL-2, IL-2, IL-3, I
L-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, I
L-11, IL-12, IL-13, IL-14, IL-15, TNF, GMC
68. The expression construct of claim 67, wherein the expression construct is selected from the group consisting of SF, β-interferon, and γ-interferon. 71. The receptor may be a CFTR, EGFR, VEGFR, I
68. The expression construct of claim 67, wherein the expression construct is selected from the group consisting of an L-2 receptor and an estrogen receptor. 72. A pharmaceutical composition comprising: (i) a first adeno-associated virus expression comprising a promoter functional in eukaryotic cells and a first polynucleotide encoding a selected polypeptide. An expression construct, wherein the first polynucleotide is under the transcriptional control of the promoter; (ii) a second polynucleotide encoding FGFR; and (ii) a pharmaceutically acceptable buffer. A composition comprising a liquid, solvent or excipient. 73. The promoter may be CMV IE, SV40 IE, R
73. The pharmaceutical composition of claim 72, wherein the pharmaceutical composition is selected from the group consisting of SV, [beta] -actin, tetracycline regulatable and ecdysone regulatable. 74. A second expression construct comprising a third polynucleotide encoding an HSPG polypeptide, wherein the third polynucleotide is operably linked to a third promoter. 8. The method of claim 7, further comprising an expression construct.
3. The composition according to 2.