CA2200869A1 - Gene therapy involving concurrent and repeated administration of adenoviruses and immunosuppressive agents - Google Patents

Abstract

A method of effecting a gene therapy treatment in a host which comprises the steps of: (a) administering to a host concurrently (i) an adenoviral vector including at least one DNA sequence encoding a therapeutic agent and (ii) an immunosuppressive agent; (b) discontinuing the administration of said adenoviral vector and said immunosuppressive agent; and (c) repeating the administration of the adenoviral vector and the immunosuppressive agent at least once. The repeated course of treatment of administration of an adenoviral vector and an immunosuppressive agent provides for continued or increased expression of the at least one DNA sequence encoding the therapeutic agent.

Description

~20g86q PATENT RULES
SECTION 104(4) NOTICE
It is the applicant's wish that, until either a patent has issued on the basis of the application or the application is refused, or is abandoned and no longer subject to reinstatement, or is withdrawn, the Commissioner only authorize the furnishing of a sample of any deposited biological material referred to in the specification to an independent expert nominated by the Commissioner in accordance with section 109 of the Patent Rules.

Feb. 3,1 997 JDM:sbf C:~l<EEP\S10-INFO.PGS

~ WO96/12406 2 2 0 0 8 6 9 PCT~S95/13253 GENE TRERAPY INVOLVING CONCURRENT AND R~PEATED
ADMINISTRATION O~ AD~NO~lKUSES AND
IMMUNOSUPPRESSIVE AGENTS
This application is a continuation-in-part of Application Serial No. 08/478,482, filed June 7, 1995, which is a continuation-in-part of Application Serial No.
08/325,679, filed October 19, 1994, the disclosures of which are incorporated by reference.
.

This invention relates to gene therapy comprising the use of adenoviruses as the gene delivery vehicles. More particularly, this invention relates tO gene therapy invoi~ing the concurrent and repeated administraticn of adenGviruses and immunosuppressive agents, wherebv the efficiency of the gene therapy treatment is enhanced through suppression of an immur.e response against the adenoviruses.

BACRGROUND OF T~E INVENTION
Adenovirus genomes are linear, double-stranded DNA
molecules of approximately 36 kilobase pairs. Each extremity of the virai genome has a short sequence :~nown as the ~nverted terminal repeat (or ITR), which is necessary for viral replication. The well-characterized molecular genetics of adenovirus render it an advantageous vector for gene transfer. Portions of the viral genome can be substituted 2 2 0 0 8 6 ~ PCT~S9S/13253 with DNA of foreign origin. In addition, recomkinant adenoviruses are structurally stable.

Adenoviruses can be very efficient in gene transfer into cells in vivo, and, thus may be employed as delivery vehicles for introducing desired genes into eukaryotic cells, whereby the adenovirus delivers such genes to eukaryotic cells by .
binding cellular receptors. There are, however, several limitations to adenovirus gene transfer which are due in part to host responses directed at either the adenovirus vector particle, breakdown products of the vector particle, or the transduced cells. These host responses include non-specific responses and specifi- lmmune responses. The non-specific responses include inflammatory and non-inflammatory changes.
An example of the latter is a change in host cell gene expression. Specific immune responses include various cellular responses and humorai antibody responses. Cellular responses include those mediated by T-helper lymphocytes, T-suppressor lymphocytes, cytotoxic T lymphocytes (CTL), and natural killer cells.

Despite the high efficiency of adenovirus vector mediated gene transfer, the translent nature of adenovirus vecto- ~ediated gene transfe- ~as suggested that repeat administrations of adenovirus ve~~ors may be necessary.
Recen. s~udies in cott^n rats however, have demonstrated that host immune responses dire~teà towards adenoviral vectors correlate wi~h decreased e ficiency of gene transfer and expression after repeated administration. Yei et ~ 7 ., Gene i~rerapy, l:l92-~Q0 (1994).

Smith, ~t al., Nature Genetics, Vol. 5, pgs. 357-402 (l9g3) discloses the administration to mice of an adenoviral vector including a human Factor IX gene. Such administration resulted in efficient liver transduction and plasma levels of WO96/12406 PCT~S95/13253 human Factor IX that would be therapeutic for hemophilia B
patients. Human Factor IX levels, however, slowly declined to baseline by nine wee~s after injection, and were not re-established by a second vector injection. Smith, et al., also found that neutralizing antibodies to adenovirus block successful repeat administration of the adenovirus.

Kozarsky, et al ., J. Biol. Chem., Vol. 269, No. 18, pgs.
13695-13702 (May 6, 1994) discloses the infusion of an adenoviral vec~or including DNA encoding the LDL receptor to rabbits. Stable expression of the LDL receptor gene was found in the rabbits for 7 to 10 days, and diminished to undetectable levels within 3 weeks. The development of neutralizing antibodies to the adenovirus resulted in a second dose being completely ineffective.

Kass-Eisler, et al ., Gene Therap~, Vol. 1, pgs. 395-402 (1994) suggest thac a T-cell response contributes to, but is not responsible solely for, the limited duration of expresslon in adul_s from adenovirus vectors. The authors further show that cyclosporin A is not effective in blocking the humoral response to the vector.
Fang, et a ., _. Cell. Biochem., Supplement 21A, C6-109, pg. 363 ;1595i d sclose the attempted re-injection of an adenovirus vector ~. dogs which were treated with cyclosporin A, an immunosuppressive agent. Such attempted re-injection was unsuccessfli.

Yang, et al . , Proc. Nat. Acad. Sci., Voi. 91, pgs. 4407-4411 (May 1994J describe recombinant adenoviruses in which the Ela and E'b regions have been deleted. ~uch viruses also include a ~ransgene. When such adenoviruses are administered to an animai hos~, cells harboring the recombinant viral genome express the transgene as desired; however, low level expression of viral genes also occurs. A virus-specific 220086q W O 96/12406 PCT~US9S/13253 cellular immune response i8 stimulated that leac1s to destruction of the genetically modified cells, thereby limiting the duration of expression of the transgene.

The aforementioned studies clearly document the need for a method of circumventing or blocking the host immune response to adenovirus vectors which prevents effective readministration o the vector, and limits the effectiveness of expression; however, they do not describe how to accomplish this.

It is therefore an object of the present invention to provide for sustained efficacy of gene transfer via repeated administration of adenoviral vectors, and for sustained expression of the transferred gene, through the suppression of an immune response against the adenoviral vectors.

BRIEF DESCRIPTION OF THE DRAWINGS
The invention now will be described with respect to the drawings, wherein:

Figure 1 is a schematic of the construction of plasmid pHR.

Figure 2 is a schematic of the construction of an expression vehicle including an adenoviral ITR, an encapsidation signal. a Rous Sarcoma '~Tirus promoter, an adenoviral triparti,e leader sequence, and linking sequences;

Figure 3 is a schematic of the construction of plasmid pAvS6;

Figure 4 is a map of plasmid pAvS6;

Figure 5 is a map of plasmid pBQ4.7;

PCT~S95/13253 Figure 6 is a map of plasmid pAvS6 - CFTR;

Figure 7 is a schematic of adenoviral vectors AvlLucl and AvlCf2;

Figure 8 is a map of plasmid pGEM-luc;

Figure 9 is a map of plasmid pAVS6-luc;

Figures lOA, lOB, and lOC depict the histologic appearance of the lung in response to AvlCf2 administration three days after vector administration;

Figures llA and llB are graphs showing the effect of dexamethasone administration on lung lavage cells at 3 days and 42 days after the administration of AvlCf2;

Figure 12 is a graph of anti-adenoviral antibody titers of lung lavage samples from rats infected with AvlCf2 and which were ~reated or not treated with dexamethasone; and Figure 13 is a graph of CTL responses in rats 42 days after infection with AvlCf2;

Fi~lre 14 is a graph of luciferase enzyme activity in ra.s infected with AvlC 2 and which were treated or no~
treated with dexamethasone, followed by infection with AvlL~Icl;

Figure 15 is a map of plasmid pAvlHgFR;

Figure 16 is a schematic of the adenovirai vector AvlH9F2;

WO96/12406 PCT~S95/13253 Figure 17 is a graph of plasma human Factor IX levels in mice which were given AvlH9F2, and were or were not given one of the immunosuppressive agents deoxyspergualin, cyclophosphamide, or dexamethasone with or without the administration of the vector AvlLac24 five weeks earlier;

Figure 18 is a graph of plasma Factor IX levels (ng/ml) in mice which received from lxlO; to lxlO~ pfu of AvlLacZ4, ~ollowed by administration of AvlH9FR five weeks later;

~ igure l9 is a graph of neutralizing antibody titer in mice that were given AvlLacZ4, and received no immunosuppression, o- were treate~ with decxyspergualin, cyclophosphamide, or dexamethasone;

Flgure 20 is a graph of plasma Factor VIII levels in mice which were g ven AvlLacZ4, and received no immunosuppression, or were given cyclophosphamide, followed by administration of AvlH9F2 with or without cyclophosphamide, followed by adm,nistratior. of AvlALAPH81;
and Figure 21 is a graph of piasma Factor IX levels (ng/ml) in mice which received AvlLacZ4 and 0 mg/kg, 5 mgJkg, lO
mg/kg, 2û mg/kg, or 3~ ~g,'kg de^~spergualir, foiiowed by administration of AvlH5F2.

DE:TAILED D~5SCRIPTION Ol~ T~E INV~NI ION
Ir accordance with an aspect of the presen_ invention, there is provided a method of e'fe-ting a gene therapy treatment in a host. Th- method comprises admlnistering to a host (i) an adenoviral vec~or ir~luding a. 'east one DNA
sequence and (ii) an immunosuppressive a~ent. T~e course of administration of tne adenovirai vector and immunosuppressive agen. then is discontinued. A~mi ni stration of the .

PCT~S95/13253 immunosuppressive agent and the adenoviral vector t~en is repeated at least once. The adenoviral vector is administered in an amount effective to produce a therapeutic effect in the host. The immunosuppressive agent is administered in an amount effective to prevent or suppress a humoral and/or cellular immune response to the vector and/or cells containing the vector.

.
The term "DNA sequence" as used herein, refers generally to a polydeoxyribonucleotide molecule and more specifically to a linear series of deoxyribonucleotides connected one to the other by phosphodiester bonds between the 3' and 5 carbons of the adjacent pentoses.

Applicants have found that, when an immunosuppressive agent is administered with the adenoviral vector, and then adminis~ration of the vector is repeated, one achieves enhanced efficacy of the repeat in vivo adenoviral-mediated gene transfer through suppression of an immune response (such as a humoral antibody response! against the adenoviral vector and/or cells transduced with the vector, and thereby achieves increased expression of the transferred genes.

The adenoviral vector which is employed may, in one embodimen_, be a. adenoviral vecto- which includes esser-ially the complete adenoviral genome. Shenk et al ., Curr. Tsp. Mlc~obiol. Immunol., 1'1 (3):l-39 (l984).
Alternat:veiy, the adenoviral vecto- may be a modified adenovira; vec~cr in which at least a portion of the adenoviral genome nas been deleted.

In another embodiment, the adenoviral vector comprises an adenoviral 5' ITR; an adenoviral 3' ITR; an adenoviral encapsidation signal; at least one DNA sequence encoding a therapeutic agent; and a promoter controlling the at least WO96/12406 PCT~S95/132S3 one DNA sequence encoding the therapeutic agent. The vector is free of at least the majority of adenoviral El and E3 DNA
sequences, but is not free of all of the E2 and E4 DNA
sequences, and DNA sequences encoding adenoviral proteins promoted by the adenoviral major late promoter.

In one embodiment, the vector also is free of at least a portion of at least one DNA sequence selected from the group consisting of the E2 and E4 DNA sequences.

In another embodiment, the vector is free of at least the majority of the adenoviral El and E3 DNA sequences, and is free of a portion of the other of the E2 and E4 DNA
sequences.

In still another embodiment, the gene in the E2a region that encodes the 72 kilodalton binding protein is mutated to produce a temperature sensitive protein that is active at 32C, the temperature at which the viral particles are produced. This temperature sensitive mutant is described in Ensinger et al., ~. Virology, 10: 328-339 (1972); Van der Vliet, et al., J. Virology, 15:348-354 (1975); and Friefeld et al., Virology, 12~:380-389 (1983~; Englehardt, et al., Proc.Nat. Acad. Sci., Vol. 91, pgs. 6196-6200 (June 1994);
Yang, et a'., Nature Genetics, Vol. ~, Dgs 362-369 (July 1994).

Such a vector, in a preferred embodiment, is constructed first by constructing, according ~o standard techniques, a shut~le plasmid which contains, beginning at the 5' end, ~he "critical left ena elemerts," which inciude an adenoviral 5' ITR, an adenoviral encapsidation slgna', and an Ela enhancer sequence; a promoter (which may be an adenoviral promoter cr a foreign promoter); a multiple cloning site (which may be as hereinabo~e described); a poly A signal; and a DNA segment WO96/12406 PCT~S95/132S3 which corresponds to a segment of the adenoviral genome. The vector also may contain a tripartite leader sequence. The DNA segment corresponding to the adenoviral genome serves as a substrate for homologous recombination with a modified or mutated adenovirus, and such sequence may encompass, for example, a segment of the adenovirus 5 genome no longer than from base 3329 to base 6246 of the genome. The plasmid may also include a .selectable marker and an origin of replication. The origin of replication may be a bacterial origin of replication. Representative examples of such shuttle plasmids include pAvS6, shown in Figure 4. A desired DNA sequence encoding a clotting factor may then be inserted into the multiple cloning site to produce a plasmid vector.

This construct is then used to produce an adenoviral vector. Homologous recombination is effected with a modified or mutated adeno~ir~s in whlch at least the ma,ority cf the El and E3 adenoviral DNA sequences have been deleted. Such homologous recombination may be effected through co-transfection of the plasmid vector and the modified adenovirus into a helper cell line, such as 293 cells, by CaPOL precipitation. Upon such homologous recombination, a recombinant adenoviral vector is formed that includes DNA
sequences derived from the shuttle plasmid be~ween the Not I
site and the homologous recombination fragmen,, and DNA
derived from the El and E3 deleted adenovirus between the homologous recombination fragment and the 3~ ITR.

In one embodiment, the homologous recombinatior.fragment overlaps with nucleotides 3329 to 6246 of tne adenovirus,5 (ATCC '~R-5) genome.

Through such homologous recombination, a ve,ctor is ~ormed which includes ar. adenoviral 5' ITR, an ader.Gviral encapsidation signal; an Ela enhancer sequence; a promoter;

_g_ 220086q WO96/12406 PCT~S95/132~3 at least one DNA sequence encoding a therapeutic agcnt; a poly A signal; adenoviral DNA free of at least the majority of the El and E3 adenoviral DNA sequences; and an adenoviral 3' ITR. The vector also may include a tripartite leader sequence. This vector may then be transfected into a helper cell line, such as the 293 helper cell line (ATCC No.
CRL1573), which will include the Ela and Elb DNA sequences, which are necessary for viral replication, and to generate infectious adenoviral particles. Transfection may take place by electroporation, calcium phosphate precipitation, microinjection, or through proteoliposomes.

The vectors hereinabove described may include a multiple cloning site to facilitate the insertion of the at least one DNA sequence encoding a therapeutic agent into the cloning vector. In general, the multiple cloning site includes "rare" restriction enzyme sites; i.e., sites which are found in eukaryotic genes at a frequency of from about one in every lO,000 to about one in every lO0,000 base pairs.
An appropriate vector in accordance with th~ present invention is thus formed by cutting the cloning vector by standard techniques at appropriate restriction sites in the multiple cloning site, and then ligating the DNA sequence enccding a therapeutic agent irto the cloning vector.

The adenoviral vector, as stated hereinabove, includes at least one DNA sequence enccding at least one therapeutic agent. The term ~herapeutic~ i 9 used in a generic sense and includes ~reating agents, prophylactic agents, and replaceme-.t agents.

~ NA seouences encoding therapeutic agents which may be placed into the adenoviral ve_tor include, but are not limited to, DNA sequences encoding tumor necrosis factor (TNF) genes, such as TNF-~; genes encoding interferons such WO96/12406 PCT~S95/13253 as Interferon-~, Interferon-~, and Interferon-~ ; genes encoding interleukins such as IL-l, IL-l~, and Interleukins 2 through 14; genes encoding GM-CSF; genes encoding adenosine deaminase, or ADA; genes encoding antioxidants such as Mn-SOD, catalase, CuZnSOD, extracellular superoxide dismutase, and glutathione reductase; genes which encode cellular growth factors, such as lymphokines, which are growth factors for lymphocytes; genes encoding gro~th factors such as epithelial growth ~actor (EGF) and keratinocyte growth factor (KGF);
genes encoding soluble CD4; Factor VIII; Factor IX; von Willebrand's factor; T-cell receptors; the LDL receptor, ApoE, ApoC, ApoAI and other genes involved in cholesterol transpor, and metabolism; the alpha-l antitrypsin (~lAT) gene, the ornithine transcarbamylase (OTC) gene, the CFTR
gene, lung surfactant protein genes, the B-glucuronidase gene, the insulin gene, negative selective markers or "suicide" genes, such as viral thymidine kinase genes, such as the Herpes Si~plex Virus thymidine kinase gene, the cytomegalovirus virus thymidine kinase gene, and the varice 'a-zoster virus thymidine kinase gene; Fc receptors for antigen-binding domains of antibodies, and antisense seouences which inhibit viral replication, such as antisense sequences which inhibit replication of hepatitis B or hepat~tis non-A non-B virus; tissue piasminogen activator ~tpA?; urinary piasminogen acti~ator (urokinase)i hirudin;
nitr~c oxide syn.hase; vasoactive pep.ides; and angiogenic peptides.

The DNA sequence encoding a therapeutic agent is under the ccntrol of a suitable promoter. Suitable promoters which may be employed include, but are not limlted to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus ( CMV?
promoter; the Rous Sarcoma Virus (RSV) promoter; inducible promoters, such as the MMTV promoter, the metallothionein 220086q PCT~S95/132~3 promoter; heat shock promoters; the albumin promoter; and the ApoAI promoter. Alternatively, the DNA sequence encoding a therapeutic agent may be under the control of its native promoter. It is to be understood, however, that the scope of the present invention is not to be limited to specific foreign genes or promoters.

Immunosuppressive agents which may be employed include those which prevent: (i) a humoral (antibody) response against the adenoviral vector; (ii) a cellular immune response, such as, for example, a T-cell response to cells containing the adenoviral vector; or (iii) a non-specific inflammatory responses against the vector and against cells containing the vector. By preventing a humoral and/or T-cell and/or non-specific inflammatory response against the vector, and/or cells containing the vector administration of the immunosuppressive agent permits effective re-administration of the vector in order to produce a therapeutic effect in the host. Preferably, where repeat administration of the adenoviral vectcr i9 desired, the immunosuppressive agent i5 an immunosuppressive agent which prevents a humoral antibody response against the adenoviral vector. Preferably, where longer duration or nigher levels of expression is desired, the immunosuppressive agent is one that prevents or suppresses a cellular sr non-specifi_ inf'ammatory response.

Host immune responses to in ~ivo adenovinls vector administration vary ir. relation to 'i) tne dose of vector;
(ii) the route of administration; ~i i! the level of replication (if any occurs); (iv) the nat~re of the transgene contained in the recombinant vector; (v! the genetic and physiological characteristics of thC host; and (vi) t;~e existence and ievei cf pre-existing immune responses to previously adminis~ered adenovirus vectors.

In general, host responses are dependent on the dose of vector administered. Importantly, the magnitude of specific 22û0869 WO96/12406 PCT~S95/13253 host responses is dependent on the route of ve-tor administration. For example, intravenous administration will yield a higher host antibody response than that of an equivalent amount of vector given via the respiratory route.
The diverse host responses to adenovirus vectors occur due to separate inflammatory and immune effector mechanisms, although most, if not all, of these distinct molecular mecha,nisms are connected and significantly interdepen~ent.
Thus, for example, humoral antibody formation is very dependent on certain T-helper lymphocyte support. Also, some cell-mediated cellular toxicity is dependent on antibody formation, e.g., opsonized macrophage cell killing.

The two principal host responses affecting the duration o~ transgene expression are the inflammatory response and the cellular immune response.

Inflammation is one of the first host responses that occurs following vector administration. Cytokine release ls very likely involved in the subsequent in lux of in~lammatory cells. Such cytokines likely include IL-1, IL-6, IL-8, and TNF .

The amount cf inflammation seen ~ollowing in vivo adenov~ral vector administration increases ~ith incresing doses cf vector given. Higher doses lead to a more rapid decline in transgene expression than do smaller doses.

CTL responses directed towards the transduced cells are believed to be important in reducins the duration of transgene expression. It is believed ~ha~ the CTL are direc~ed against low level adenovirus ger.e expression by the cells, which induces the CTL.

The principal host response affecting the ability to administer adenovirus vectors repeatedly to a host is the humoral antibody response. It develops tc adenovirus 220086q WO96/12406 PCT~S95/132~3 administered by a variety of routes, including oral, intravenous, intraperltoneal, and intrapulmonary. In general, the level of antibody response achieved i5 very dependent on the dose of vector administered. The antibody response is also dependent on the route of vector a~i n; stration. Intravenous vector administration results in higher antibody levels than pulmonary administration for a given dose of vector. In contrast, wild type adenovirus elicits high antibody levels irrespective of the amount of virus given due to virus replication in vivo. The ability to repeat successfully adenovirus vector administration is inversely correlated with the level of circulating anti-adenovirus vector antibody present.

Pharmacologic modulation of host immune responses to adenovirus vectors involves the use o' anti-inflammatory agents, celllular immune modifiers, and humoral antibody immune modifiers.

An.i-inflammatory agents include steroids, cyclophosphamide, and azothiophrine.

Steroids have potent anti-inflammatory properties.
Applicants have showr. that steroids, such as dexamethasone, given parenterally prolong th- durar i5n of transgene expression following in vivo adm nistratlon or vector~ia the lung route. Steroids also bloc~ the function of lymphocytes.
Thus, dexamethasone reduces the CTL responses observed after pulmonary vector administratior. Dexamethasone also blocks, at ieast in part, the host antibody response to adenovirus vec-or administration.

Antibodies directed at -ellular components of the immune system reduce cellular immune response. For example, anti T-cell receptor antibody (such as, for example anti-CD4 and anti-CD3 antibodies) administration prolongs transgene expression. CTLA4 immunoglobulin is another example. Anti-220086~
WO96112406 PCT~S95/13253 CD4 antibody is directed against the T-helper lymphocytes and reduces their function. Other agents directed primarily at the cellular immune response include cyclosporins such as cyclosporin A; rapamycin binding protein ligands such as FK506; and steroids such as dexamethasone.
Agents which affect humoral antibody responses are generally directed at antibody producing B lymphocytes (B-cells) or at the T-cells which are responsible for in~ucing B-cell antibody production to high levels.

Examples of immunosuppressive agents which prevent a humoral antibody response against the adenoviral vector include, but are not limited to, deoxyspergualin, or DSG, which has the following structure:
H.NCNH(CH~)lCH7CH.CONHCHCONH(CH2)lNH(CHJ)3NH, NH OH
The terms "deoxyspergualin and "DSG" as used herein, mean deoxyspergualin or DSG and derivatives or analogues therecf, such as salts of deoxyspergualin, including but not limited tO , trihydrochlorides thereof, and any other analogues which have immunosuppressive activity. Such compounds are described further in U.S. Patent Nos.

4,525,259; 4,817,299; 5,162,581; and 5,196,453.

In o..- embodiment, the immunosuppressive agent which prevents a r,umoral antibody response is a steroid. Steroids which may be employed include, but are nol limited to, dexamethasone, and any adrenocortical hormones, such as,for example corticosteroidsi hydrocortisone; prednisolone; and methylpre~n solone.

In another embodimen~, the immunosuppressive agent which prevents a humoral antibody response is a cyclosporin, such as, for example, cyclosporin A. Other immunosuppressive agents which prevent a humoral antibody response and which may be employed include, but are not limited to, 220086~
WO96/12406 PCT~S9S/132~3 azathioprine; cyclophosphamide; brequinar; leflunGmide;
mycophenolate mofetil; anti-CD40 antibodyi anti-CD40 ligand antibody; cyclophosphamine; rapamycin; anti-CD4 antibody;
CTLA-4 immunoglobulin; Interleukin -12; Interferon -~;
rapamycin binding protein (FEBP) ligands, such as, for example, FK506, as described in Bierer, et al., Proc. Nat.
Acad. Sci., Vol. 87, pgs. 9231-9235 (1990); Dumont, et al., J. Immunol., Vol. 144, pgs. 1418-1424 (1990); and Bierer, et al., Science, Vol. 250, pgs. 556-559 ~1990); anti-lymphocyte function antigen-l (LFA-l) antibody; and anti-T-cell receptor antibody.

Applicants have found that, when compounds which prevent, suppress, or eliminate humoral immune responses to foreign antigens (such as, for example, deoxyspergualin, cyclophosphamide, brequinar, leflunomide, mycophenolate, mofetil, anti-CD40 antibody, or anti-CD40 ligand antibody) are administered at a short time prior to, and/or during, and/or for a short time after adenoviral vector administration, to a host, such compounds prevent the production of anti-adenoviral neutralizing antibodies in the host. The prevention of the production of such neutralizing antibodies enables the efficient re-administration of the adenoviral vector to the host.

It is to be understood that, withln the scope of the present invention, that an immunosuppressive agent may prevent more than one of the immune responses hereinabove described. It also is to be understood, however, that the scope of the present invention is not intended to be limited to any specific immunosuppressive agents.
.

It is also contemplated that within the scope of the present invention, a combination of immunosuppressive agents may be employed.

2 2 ~ q ~ l WO96/12406 PCT~S95/13253 The adenoviral vector and immunosuppressive agent, in general, are administered concurrently in an amount effective to produce a therapeutic effect in the host while preventing an immune response against the vector or against cells transduced with the vector. The term "concurrently," as used herein, means that the administration of the adenoviral vector and administration of the immunosuppressive agent are begun at approximately the same time, i.e., within a brief time frame of each other, and the administration of the adenoviral vector and the administration of the immunosuppressive agent are parts of a unitary course of treatment. Thus, for example, the immunosuppressive agent is administered at approximately the same time the adenoviral vector is administered, i.e., the administration of the immunosuppressive agent is begun at a short time (for example, about 24 hours) before, or during, or at a short time (e.g., 24 hours) after the administration of the adenoviral vector. In general, the ;mmllnosuppressive agent is administered according to standard dosage schedules established for that agent, and for a period of time which in general does not exceed 14 days, and preferably does not exceed ll days, and more preferably does not exceed 8 days.
Thus, long-term administration of the immunosuppressive agent is not required for enabling repeated administration of the adenovirus.

At the conclusion of the course of administration of the immunosuppressive agent, the course of administration of the adenoviral vector and immunosuppressive agent is discontinued for a period of time. The period of time between courses of administration of the adenoviral vector and the immunosuppressive agent, and the number of courses of administration of the adenoviral vector and immunosuppressive agent is dependent upon a variety of factors, including the age, weight, and sex of the patient, the disease or disorder SUBSTITUTE SHEET (RULE 26) 220086q WO96/12406 PCT~S95/13253 being treated, and the severity of the disease or disorder being treated.

It is to be understood that the above course of administration of the immunosuppressive agent is repeated with each administration of the adenoviral vector.

In one embodiment, the adenoviral vector may be administered, at each administration, in an amount of from l plaque forming unit ~o about lO;' plaque forming units, preferably from about lO~ plaque forming units to about lo;t plaque forming units, more preferably from about lO~ to about lOI~ plaque forming units per kg. The host may be a human or non-human animal host.

The adenoviral vector may be administered systemically or topically. Examples of systemic administration include, but are not limited to, intravenous administration (such as for example, portal vein injection or peripheral vein injec~ion), intramuscuiar administration, intraperitoneai administration, intranasal administration, or encapsulated oral administration.

The immunosuppressive agent is ad~._r.is.ered in an amo~n~
effective to produce a desired immunosuppressive effec~ in tne host. The immunosuppressive agent may be administere~, at eacn administration, in an amount cf from about l ~gjkg -o about 15 mg/kg, when dexamethason- is employed, or at ~ne dose equivalents for other steroids. When deoxyspergualln is employed, the deoxyspergualin may be administered in an amoun_ of from about l mg/kg to about 33 mg/kg, preferabiy from about 3 mg/kg to about 7 mg/ka. When cyclophcsphamide is employed, the cyclophopham1de may be administered in an amount of from about 5 mg/kg to about 300 mg/kg, preferably from about 50 mg/kg to about lO0 mg/kg.

WO96/12406 PCT~S95/132S3 The adenoviral vector particles and the immunosuppressive agent each may be administered in combination with a pharmaceutically acceptable carrier suitable for administration to a patient. The carrier may be a liquid carrier such as, for example, a saline solution.
The adenoviral vector particles also may be administered in combination with a solid carrier, such as, for example, microcarrier beads, or a sustained drug delivery material, such as, for example, a polyol.

Cells which may be transduced by the adenoviral particles include, but are not limited to, lung, airway, or alveolar epithelial cells; primary cells, such as primary nucleated blood cells, such as leukocytes, granulocytes, monocytes, macrophages, lymphocytes (including T-lymphocytes and B-lymphocytes), totipotent stem celis, and tumor infiltrating lymphocytes (TIL cells); bone marrow cells;
endothelial cells; activated endothelial cells; epithelial cells; keratinocytesi stem cells; hepatocytes, including hepatocyte precursor cells; fibroblasts; mesenchymal cells;
mesothelial cells; parenchymal cells; vascular smooth muscle cells; brain cells and other neural cells; gut enterocytes;
gut stem cells; and myoblasts.

In one embodimen~, the adenovira pa~ti_les may be targeted tO blood ceils, whereby such adenoviral vector particles infect the blood cells with a gene which directly or ir.directly enhances the therapeutic effects of the blood cells. The gene carried by the blood cells can be any gene which allows the blood cells to exert a therapeutic efrect that it would not ordinarily have, such as a gene encoding a clotting factor useful ir the treatment of hemophilia. The gene can encode one or more products having therapeutic effects. Examples of suitable genes include those that encode the CFT~ gene; cytokines such as TNF, interleukins 2 2 0 0 8 6 q PCT~S95/13253 (interleukins 1-14), interferons (~, B, ~-interferons), T-cell receptor proteins and Fc receptors for antigen-binding domains of antibodies, such as immunoglobulins. Other examples of suitable genes include genes encoding soluble CD4 which is used in the treatment of AIDS and genes encoding ~-antitrypsin, which is useful in the treatment of emphysema caused by ~-antitrypsin deficiency.
, The transduced cells are useful in the treatment of a variety of diseases including but not limited to, cystic fibrosis, adenosine de~m;n~.ce deficiency, sickle cell anemia, thalassemia, hemophilia, diabetes, ~-antitrypsin deficiency, brain disorders such as Alzheimer's disease, phenylketonuria and other illnesses such as growth disorders and heart diseases, for example, those caused by alterations in the way cholesterol is metabolized and defects of the immune system.

In another emDodiment, the adenoviral vector particles may transduce liver cells, and Suc:rL adenoviral vector particles may include gene(s) encoding polypeptides or proteins which are useful in preventicn and therapy of an acquired or ar inherited defect in hepatocyte (liver) function. For example, they can be used to correct an inhe_iteà de~iciency of the low densi~-v l poprotein (T DL) receptor, and/or to correct an inherited dericiency of ornithine transcarbamylase (OTC), which results in eongenital hyperam~.onemia.

Ir. another embodimen., the adenovira_ parti_les may transduce liver cells, whereby tne adenoviral particles include a gene encoding a therapeutic agent emp'oyed to treat acquired infectiQus diseases, such as diseases resulting from viral infection. For example, the infectious adenoviral particles may be employed to treat viral hepatitis, 220~6~
WO96/12406 PCT~S95/132~3 particularly hepatitis B or non-A non-B hepatitis. For example, an infectious adenoviral particle containing a gene encoding an antisense gene could be employed to infect liver cells to inhibit viral replication. In this case, the infectious adenoviral particle, which includes a vector including a structural hepatitis gene in the reverse or opposite orientation, would be introduced into liver cells, resulting in production in the infected liver cells of an anti-sense gene capable of inactivating the hepatitis virus or its RNA transcripts. Alternatively, the liver cells may be infected with an infectious adenoviral particle which includes a gene which encodes a protein, such as, for example, ~-interferon, which may confer resistance to the hepatitis virus.

The vector particles also may be employed in treating Hodgkin's lymphoma. An infectious adenoviral vector particle may be targeted to neoplastic cells of Hodgkin's lymphoma.
The adenoviral vector particle also includes a negative selective marker or "su cide gene, such as the Herpes Simplex thy~idine kinase gene. The adenovirus may be administered ln vivo tG a patient, whereby the virus infects neoplastic cells of Hodgkin's lymphoma. After the adenovirus is administered to the patient, the patient is given an interaction agen.
such as gancyclovir or acyclovir, whereby the neoplastic Hodgkin's lymphoma cells infected with the adenovir~s are killed.

In addition, a vector may be constructed which includes the CFLR gene. The vector then may b^ administered to the respiratory epithelium in an effective therapeutic amount for the correction of the pulmonary defici~ in patients with cysti^ fi~rosis. In another example, vectors containing functional proteins may be delivered to the respiratory epithelium in order to correct deficiencies in such proteins.

WO96/12406 2 2 0 0 8 6 9 PCT~S9S/13253 ~
Such functional proteins include antioxidants, antitrypsin, CFTR, lung surfactant proteins, cytokines, and growth factors such as BGF and KGF, and may also include adenosine deaminase for treatment of severe combined immune de~iciency, von Willebrand's ~actor for treatment of Christmas disease, and ~-glucuronidase for treatment of Gaucher's disease. Also, vectors including genes encoding anti-cancer agents or anti-inflammatory agents may be administered to lung cells of a patient for the treatment of lung cancer or inflammatory lung disease.

EXAMPLES
The invention now will be described with respect to the following examples; however, the scope of the present invention is not intended to be iimited thereby.

Exam~le l Construction of AvlCf2 and AvlLucl A. C~nstruction of PAvS5 The adenoviral construction shuttle plasmid pA~S5 was constructed in several steps using standard cloning techniques including polymerase chain reaction based cloning techniques. First, the 2913 bp BglII, HindIII fragment was removed from Ad-dl327 and inserted as a blurt fragment into the XhoI site of pBluescript II KS-(Stra~gene, La Jolla, C~) (Figure l).

Ad-dl327 is identical to adenovirus 5 except .hat an XbaI ~ragment including bases 28591 to 33474 (or map units 78.5 to 84.7) of the Adenovirus 5 ge.Lome, and which is located in the E3 region, has been deieted. The E3 deletion in Ad-dl327 is similar to the E3 deletion in Ad-dl324, wh ch is described in Thimmappaya et al., Celi, 3i:543 (1983). The complete Adeno~irus 5 genome is registered as Genba.~k accession #M73260, incorporated herein by rererence, and the .

WO96/12406 PCT~S95/13253 virus is available from the American Type Culture Collec'ion, Rockville, Maryland, U.S.A. under accession number VR-5.

Ad-dl327 was constructed by routine methods from Adenovirus 5 (Ad5). The method is outlined briefly as follows and previously described by Jones and Shenk, Cell 13:181-188, (1978). AdS DNA is isolated by proteolytic digestion of the virion and partially cleaved with Xba I
restriction endonuclease. The Xba I fragments are then reassembled by ligation as a mixture of fragments. This results in some ligated genomes with a sequence similar to Ad5, except excluding sequences 28591 bp to 30474 bp. This DNA is then transfected into suitable cells (e.g. KB cells, HeLa celis, 293 cells) and overlaid with soft agar to allow plaque formation. Individual plaques are then isolated, amplified, and screened for the absence of the 1878 bp E3 region Xba fragment.

The orientation of this fragment was such that the BglII
site was nearest the T7 RNA polymerase si~e of pBluescript II
KS- and the HindIII site was nearest the T3 RNA polymerase site cf pBluescript II KS-. This plasmid was designated PHR.
~Figure 1).

Second, the ITR, e~capsidation signa', Rous Sarcoma Virus promoter, the adenoviral ~r~partite leader (TPL) sequence and linking sequences were asse~bled as a block using PCR amplification (Figure 2). The ITR and encapsidation signal (sec~ences 1-392 of Ad-dl327 [identical to sequences from AdS, GenbanX accession #M73260]
incorporated herein by reference ! were amplified (amplification 1~ together from Ad-dl327 using primers containing NotI or AscI ~estriction sites. The Rous Sarcoma Virus LTR promoter was amplified (amplification 2) from the plasmid pRC/RSV (sequences 209 to 605; Invitrogen, San Diego, ~ 2200869 WO96/12406 PCT~S95/13253 CA) using primers containing an AscI site and an SfiI site.
DNA products from amplifications 1 and 2 were joined using the "overlap" PCR method (amplification 3) (Horton et al ., BioTechni~ues, 8:528-535 (1990)) with only the NotI primer and the SfiI primer. Complementarity between the A~cI-containing end of each initial DNA amplification product from reactions 1 and 2 allowed joining of these two pieces during amplification. Next the TPL was amplified (amplification 4) (sequences 6049 to 9730 of Ad-dl327 [identical to similar sequences from AdS, Genbank accession #M73260]) from cDNA
made from mRNA isolated from 293 cells (ATCC Accession No.
CRL 1573) infected for 16 hrs. with Ad-dl327 using primers containing SfiI and XbaI sites respectively. DNA fragments from amplification reactions 3 and 4 were then joined using PCR (amplification 5) with the NotI and XbaI primers, thus creating the complete gene biock.

Third, the ITR-encapsidation signal-TPL fragment was then purified, cleaved with NotI and XbaI and inserted into the NotL, XbaI cleaved PHR plasmid. This plasmid was designated pAvS6A- and the orientation was such that the NotI
site of the fragment was next to the T7 RNA polymerase site (Figure 3).

Fourtn, the SV40 early polyA signal was removed from SV40 DNA as an HpaI-BamHI fragment, treated with T4 DNA
pol~merase and inserted into the Sall site of the plasmià
pAvS6a-;Figure 3) to create pAvS6 (Figures 3 and 4).

B. Construction of AvlCf2 and AvlLucl Av'Cf2 tFigure 7) (Yei et al., Gene Therapy, 1:192-20C
(1994)), an E1-deleted (i.18 map units ~o 9.2 map units), E3-deleted (78.5 map units to 84.7 map units) adenoviral vector constructed first by inserting the normal human CFTR cDNA
coding sequence fragment into the EcoRV si~e of pAvS6 so that 2 2 0 0 8 6 9 PCT/USg5/13253 the 5' end of the CFTR coding sequence was closest .o the Adenovirus 5 tripartite leader- The CFTR cDNA was removed as a PstI fragment (nucleotides 75 to 4,725; for numbering see GenBank Accession No- M28688) from the plasmid pBQ4.7 (Figure 5) (provided by L.-C- Tsui, The Hospital for Sick Children, Toronto, Canada), and inserted as a blunt fragment. The resulting plasmid, pAvS6-CFTR (Figure 6) was linearized with KpnI and recombined with the large (35 kb) ClaI fraglnent of Ad-dl327 in 293 cells as described in Trapnell, Advanced Drug Delivery Reviews, 12:185-199 (1993) to form AvlCf2 (Figure 7) .

After double-plaque purification, the identify of the clonal isolates was confirmed by Southern analysis, immunoprecipitation of CFTR, as prevlously described.
(Tolstoshev, et al., Proceedings of the Ninth Nagoya International Symposium on Cancer Treatment, September 17-18, 1993, Nagoya, Japan (in press).

AvlLucl (Figure 7) (Yei e~ al., Gene ~herapy, Vol. 1, pgs. 192-200 (1994) ) is an adenoviral reporter vector identical in genomic organization and sequence to AvlCf2, except that it expresses the firefly luciferase gene (Genbank Accession No. M15077).

The firefly luciferase gene was obtained from pGEM-luc (Figure 8 - Promega! pGEM-luc was digested with StuI and HindIII in order to splice out the firefly luciferase gene.

The firefly luciferase gene was inserted into the EcoRV si~e of pAvS6 so that the 5' end of the firefly luciferase coding sequence was closest to the Adenovirus 5 tripartite leader- The resulting plasmid, pAvS6-Lucl (Figure 9) was linearized with KpnI and recombined with the large (35 kb) ClaI fragment of Ad-dl327 as hereinabove described.

.~. 2200869 6 PCT~S95/13253 Clonal isolates then were identified as herei~above described.

Both viral vectors were propagated, purified by double-banding in CsCl gradients~ and titered in 293 cells as described in Rosenfeld et al ., Cell, 68:143-155 (1992).

ExamPle 2 Adenoviral-mediated qene transfer with concurrent intermittent steroid administration Cotton rats (weight approximately 150g) were divided into four groups with 9 rats in each group. AvlCf2 was administered by intranasal inhalation (Yei et al., Human Gene Therapy, Vol. 5, pgs. 731-744 (1994)) to the lungs of cotton rats at a low dose (10~ pfu) or at a high dose (101 pfu), either with or without coadministration of dexamethasone by intraperitoneal injection in an amount of 2 mg/kg daily, beginning 1 day prior to and continuing for 10 days after administration of the vector. A control group of rats was given PB~ ir.stead of AvlCf2, either w~th or without coadministration of dexamethasone as hereinabove described.

At 3 days and at 42 days after vector administration, 3 rats from each group were evaluated fo~ hosr responses to the AvlCC2 vector. Evaluations sf hos. responses included pulmonary histopathology appearance, ~o-al lung lavage cellularity, lung lavage anti-adenovirus antibody production, and cytotoxic .-lymphocyte (CTL' response.

Evaluation of pulmonary histopathoio~y was performed as described -n Yei et al., Human Gene ~herapy, Vol. 5, pgs 731-744 (1994j. The lungs were removed, fixed in 2.5~ (wt./vol.`~
paraformaldehyde, 0.25~ (wt./vo' . ~ giutaraldehyde al 4C
overnight, embedded in paraffin, and 6~m sections were stained with hematoxylin and eosin.

220086~ ^
WO96/12406 PCT~S95/13253 The histologic appearance of the lungs of the ~otton rats in response to AvlCf2 injection, with and without daily administration of immunosuppression therapy, at three days after adenoviral infection, is shown in Figures lOA, lOB, and lOC. Figure lOa is a section of the lung of a control rat that did not receive adenovirus and instead received PBS.
Figure lOB is a section of the lung of a rat which received AvlCf2 without immunosuppression therapy. Figure 10- is a section of the lung of a rat which received AvlCf2 with daily administration of dexamethasone. All sections have been magnified 100 times. As shown in Figures lOA, lOB, and lOC, there was less pulmonary parenchymal inflammation in the rat which received adenovirus and immunosuppressive therapy as compared with the control rat and the rat infected with adenovirus, but did not receive immunosuppression therapy.

Lung lavage fluid was collected by lavaging the lung with 4.0 ml of PBS, and the total number of cells determined by counting in a hemocytometer, or the cells were evaluated in cytocentrifuge preparations for the percentage of neutrophils by light microscopy.

Figure llA is a graph cf the lung lavage cell count from rats infected with AvlCf2 three days after infection, as compared with con~rcl ra.s which rece~ved PBS. The control rats either received dexamethasone or did not receive dexamethasone. Figure llB is a grapn of the lung lavage cell coun~ from rats infected with AvlCf2 at 42 days after infection, as compared w~h control rats which received PBS.
The rats either receivea dexamethasone ~r did not receive immunosuppressant therapy.

As shown in Figure llA, dexamethasone significantly reduced the non-specific host cellular inflammatory responses (represented by total lung lavage cellularity) at three days - 220086~
WO96/12406 PCT~S95/13253 after vector administration, which is the peak of inflammation.

Lung lavage anti-adenovirus production was measured by an ELISA assay carried out as follows.

10~1 of AvlLacZ4 (Yei, et al., Human Gene Therapy, Vol.
5, pgs. 731-744 (1994)) at lxlOIl pfu/ml was added to 9~ ~l of double distilled H~0 in a 0.5 ml Eppendorf tube. The tube was irradiated with ultraviolet light for 30 minutes in order to kill the adenovirus, and protein concentration was measured with a Bio-Rad kit. 8 ml of O.lM Na.C03 (pH 9.6) was added to the tube to provide a protein yield of 10 ~g/4 ml or 125 ng of adenoviral antigen per 50 ~l per well.

50~1 of antigen then was added to each well of a 96-well microtiter plate (Immulon 2); and the plate was incubated at 37C for 1 hour, or room temperature for 2 hours, or at 4C
overnight. The plate then was washed twice with PBS or doub_e disti;ied HØ

300~1 of blocking buffer (1~ BSA in PBS) was added to each well, and the plate was incubated for ' hour at room temperature. The plate then was rinsed wi~h ~ouble distilled H 0.

Blocking agent then was added to the background wells.
5C~l of antibody samples (i.e., lung lavage samples prepared as hereinabove described) then were added to ccated wells at seria' two-fold dilutions, beginning at 1/1 and ending at 1/8192. 50 !~1 of negative control sampies of serum from an uninfected cotton rat were added to another set of coated welis at the same serial dilutions. The plate the was inc~bated for 2 hours at room temperature, and 300 ~l of 0.05~ Tween 20/PBS then was added. The plate was incubated 22 00869 - ~
WO96/12406 PCT~S95113253 for 5 minutes at room temperature, emptied, and 300~1 of O.05~ Tween 20/PBS again was added. The plate again was incubated at room temperature for 5 minutes, and wa~ emptied.
The plate then was washed twice with double distilled H~0 or PBS.

Peroxidase-labeled goat anti-hamster IgG (l0~g/l0~l) was diluted with l0 ml BSA and PBS to make a working solution of l mg/ml concentration. (l:l,000 dilution). l00 ~l of this solution then was added to each well, and the plate was incubated at room temperature for 2 hours. The plate then was washed five times with 300 ~l of 0.0l~ Tween 20/PBS. The plate then was emptied and dried.

100 ~1 of tetramethyl benzidine (TM3) substrate was added to each well at room temperate, and the color was developed immediately. The blue color was monitored by reading at ODo~. with an ELISA reader. The reaction was stopped when the OD~,o was 0.5 to 0.6 by adding l00 ~l of TMB
sto~ solution to the wells. The ODI~, then was read betwee..
5 minutes and l hour after stopping the reaction. The antibody titer is the reciprocal of the dilution that gave an OD value of 0.l larger than background OD. Alternatively, antibody titer also may be determined as 3 standard devia ions above the OD of non-specifi- ackground. Th~
average results, expressed in antibody titer, for the rats in each group at 3 days and 42 days after vector administration are shown in Figure 12. As shown in Figure 12, the rats which we~e given dexamethasone showed a decreased antibody titer ~2 days after vector administration as compared with rats ~hat we~e not given dexamethasone.

C~' assays were carried out at 42 days after vector administration.

WO96/12406 PCT~S95/13253 Sensitizer cells were prepared by infecting cotton rat lung fibroblasts with Ad-dl327 at a multiplicity of infection of lO0. The cells were incubated for 3 days, and checked for hexon expression by FACS. The cells then were washed with PBS/EDTA, contacted with trypsin, washed, spun, and resuspended in l ml RPMI medium. The cells then were irradiated with 13 Cs at 5,000 rads in order to inactivate the DNA.

Spleens then were isolated from uninfected (control) rats and adenovirus-infected rats 42 days after infection.
The spleens were kept in sterile HBSS and ice. lO ml of HBSS
then was injected into each spleen with a 25/27 gauge needle.
The spleen was mashed, and filtered with a cell strainer into a 50 ml tube. The ~olume then was brought to 40 ml in RPMI
plus lO~ FCS. The tube was spun at l,500 rpm for lO minutes.
Red blood cells then were lysed by adding 2.5 ml of ACK lysis buffer, and the liquid was swirled for less than l minute.
The volume was brought up to 53 ml with RPMI-lO. The tube then was spun again at l,500 rpm for 0 m nutes. The cell pellet then was resuspended, and celis were counted at a l:lO
dilution. The splenocytes then were plated with the sensitizer cells at a ratio of splenocytes to sensitizer cells of 4:l in ~PMI. The splenocytes and sensitizer cells were incubated at 37C in th~ presence Or 20-50 un tsi~; of Interleukin-2. In~erleukin-2 was adde~ daily for 5 ~o days.

Target cells were prepared by in~ecring 3XlO~ cotton rat lung fibroblasts with Ad-dl327 at a multiplicily of infectior of lO0 for l hour. ~ulture medium is added to tne cells, and `iCr in an amount greater than 50 ~Ci is adde ~or 18 hours.

Target cells are harvested by washing the cotton ra~
lung fibroblasts with EDTA/PBS, followed by trypsinization.

220086q WO96/12406 PCT~S95/13253 The cells then were washed, spun, resuspended in 5 ml culture medium, and counted. The cells were resuspended to 105 cells/ml and 10~ cells/0.1 ml well were used for the CTL
assay.

Effector cells (i.e., the combination of splenocytes and sensitizing cells (also sometimes referred to as Es cells) were spun at 1,500 rpm for 10 minutes at 4C. The ce ls were resuspended in 2 ml of HBSS-10, loaded onto 7 ml Ficoll Hypaque, and spun at 1,500 rpm for 10 minutes. The top portion (4 ml) was harvested, and 5 ml of culture medium was added. This material was spun, the cell pellet was saved, and resuspended in 1 ml of culture medium. The effector cells were counted by mixing 50 ~l of effector cells with 50 ~1 Trypan blue.

The effector cells then were added to wells containing 10' target cells, at effector:target (E:T) ratios of 3.125, 6.25, 12.5, 25, 50, and 100. The cells then were spun at 500 rpm for 5 mlnutes. The ceiis t~en were incubated at 37C for 4 hours. The cells then were spun, and 100 ~l of supernatant was analyzed for ~icr release with a WALLAC gamma counter.
The average results for CTB response in splenocytes taken ~rom in~ected rats (with and without dexamethasone _reatment), and frcm tws un_r.fected controi rats, are shown in Figure l3.

As shown in Figure 13, a lower CTL response was obtained from splenocytes obtained from infected rats that were treated with dexamethasone.

A~ 42 days after vector aaministration, the remaining three rats in each gro ? received an intrar.asal pulmona~y administration of AvlLu_1 at a dose of 2xlO pfu. Remaining control rats, which initially received PBS, also received 220086q WO96/12406 PCT~S95/132~3 AvlLucl at a dose of 2xlO9 pfu. Lung lavage anti-adenovirus antibody production was evaluated three days after administration of AvlLucl, according to the procedure hereinabove described, and the results are shown in Figure 12. As shown in Figure 12, the rats which initially were given 10l~ pfu of AvlCf2, and were treated with dexamethasone, showed a decreased antibody titer as compared with rats that were not given dexamethasone.

The efficacy of transfer and expression of the firefly luciferase gene was evaluated three days after administration by quantifying directly intrapulmonary luciferase enzyme activity in light units (lu) by routine luminometry as described in Yei et al ., Gene Therapy, Vol . 1 , pgs 192-200 (1994). The results are shown in Figure 14. As shown in Figure 14, each dot represents the luciferase enzyme activity for one rat, and the cross-bar represents the mean for each group. The efficiency of repeat adenovirus-mediated gene transfer was significantly higher in the rats which received AvlCf2 ar.d dexamethasone than those which did not receive dexamethasone at the time of the first adenoviral administration (11,786 + 3523 lu vs. 622 + 192 lu, respectively). The efficiency of gene transfer from AvlLucl also was higher in the control group which initially received PBS in conjunction with dexamethasone.

ExamPle 3 Suppression of humoral immune response with DS~ or hiqh dose cyclophos~hamide, Permittinq effec-ive re~eat administration of an adenoviral vector This example describes the intravenous administration of the adenoviral vectors AvlLacZ4 and AvlH9F2 to C57BL/5 ma'e mice (Harian Sprague Dawley, Indianapolis, Indiana) at 5 to 6 weeks of age at the start of the experimen.. AvlLacZ4 is an adenoviral vector which includes a nuclear targeted B-~ WO96/12406 2 2 0 0 ~ 6 ~ PCT~S95/132~3 _ galactosidase gene, (lacZ) and is described in PCT
application No. W095/09654, published April 13, 1995.
AvlH9F2 is constructed from a derivative of the adenoviral shuttle plasmid vector pAvlH9FR (Figure 15), which includes human Factor IX DNA, and is described in PCT application No.
W094/29471, published December 22, 1994.
. .
To construct AvlH9F2, the shuttle plasmid pAvlH9FR was digested with the restriction enzyme Sfil, the-DNA ends were made blunt using T4 DNA Polymerase, and the DNA molecule was recircularized by ligation. Competent DH5 cells were transformed and ampicillin-resistant clones were amplified and screened by restriction enzyme digestion of miniprep DNA.
A positive clone was identified and the resulting shuttle plasmid was referred to as pAvS17H9F.

Subsequently, 293 cells were cotransfected with pAvS17H9F and the large DNA fragment of ClaI digested Ad-dl327. Recombinant adenoviral vector plaques were picked, expanded, and screened for expression of Factor IX by ELISA.
A positive clone was identified and amplified, thus generating the vector AvlH9F2. A schematic of the left end of the vector is shown in Figure 16. AvlH9F2 has a base pair deletion at the beginning of the tripartite leader, or TPL, which effectively changes the ATG into a CTG. The structure of the vector was verified by restriction enzyme diagnostics and by DNA sequence analysis of the region between the RSV
promoter and the 3' untranslated region of the Factor IX
cDNA.

Fifteen mice were ;mmllnosuppressed with 33 mg/kg of deoxyspergualin DSG (Nippon Kayaku Co. LTD, Tokyo, Japan), delivered intraperitoneally (ip), once daily, beginning the day before vector administration and continuing for a`total of eight days. A vial containing 100 mg of lyophilized DSG

SUBSTITUTE SHEET (RULE 26) 220086q PCT~S95/13253 was reconstituted with 3.8 ml of injection grade water to yield a 25 mg/ml solution, which was aliquoted and frozen at -20C. Each day, immediately before imml~nosuppression, an aliquot was thawed at room temperature and 0.7 ml wa~ mixed with 6.3 ml of Hank's Balanced Salt Solution (B SS) to yield a 2.5 mg/ml solution. The mice were weighed once, immediately prior to the first dose of DSG. Six mice received l x lO~ pfu of AvllacZ4 via tail vein injection on the second day of the immunosuppression regimen and l x 108 pfu of AvlH9F2 five weeks later. Another six mice received only A~lH9F2, five weeks after immunosuppression. Three mice were immunosuppressed, but received no adenoviral vector.

Six mice were immunosuppressed with a low dose (lO0 mg/kg) cyclophosphamide (Sigma) and fifteen mice were treated with a high dose (300 mg/kg). The animals received a single ip injection of cyclophosphamide one day before administration of adenoviral vector. All six mice which were treated with a low dose of cyclophosphamide also received l x lC~ pfu of AvlIacZ4 the day after cyclophosphamide and l x lOd pfu of AvlH9F2 five weeks later. Six of the mice immunosuppressed with a high dose of cyclophosphamide received a l x lO~ pfu of AvllacZ4 the next day and l x lO~
pfu of AvlH9F2 five weeks later. Another six did not receive AvllacZ4 but did receive AvlH9F2. Finally, three mice were immunosuppressed, but received no adenoviral vector.

~ elve mice were immunosuppressed with 5 mg/kg dexamethasone (American Reagent Laboratories, Inc., Shirley, New York), delivered ip, once daily, Deginnlng the day ~efore vector adminisrration and continuing for a total of eight days. S x of these mice receive l x lO~ pfu of AvllacZ4 on the second d~y of dexamethasone treatment and l x lQ~ Ffu of AvlH9F2 five weeks later. Five immunosuppressed mice received only AvlH9F2 and one mouse received no vector.

WO96/12406 PCT~S95/13253 -~v Five weeks after administration of AvllacZ4, but prior to ~m; n; stration of AvlH9F2, plasma was prepared from some mice and analyzed for antiadenovirus neutralizing antibodies.
Neutralizing antibodies were detected in the plasma of mice which receive AvlLacZ4 without ;mmllnosuppression, however, mice which received vector and either DSG or high dose cyclophosphamide had no detectable neutralizing antibodies.
In contrast, mice ;mml~nosuppressed with low dose cyclophosphamide or dexamethasone developed neutralizing antibodies.

Neutralizing antibody titers in 20 mice are given in Table I below. As indicated in Table I, DSG is deoxyspergualin, Cy is cyclophosphamide, and Dex is methasone.
Table I
mouse vector ;mmllnosuppression neutralizing Ab l none none 0 2 none none 0 3 lxlO8 pfu none 64 AvllacZ4 4 lxlO8 pfu none 32 AvllacZ4 lxlO8 pfu none 32 AvllacZ4 6 lxlO8 pfu none 32 AvllacZ4 7 none 33 mg/kg DSG o 8 none 33 mg/kg DSG o 9 lxlO8 pfu 33 mg/kg DSG 0 AvllacZ4 lO lxlO8 pfu 33 mg/kg DSG 0 AvllacZ4 ll none 300 mg/kg Cy o 12 none 300 mg/kg Cy 0 SUBSTITUTE SHEET (RULE 26) 220086~
WO96/12406 . PCT~S95/132S3 13lx108 pfu 100 mg/kg Cy 64 AvllacZ4 14lx10~ pfu 100 mg/kg ~y 8 AvllacZ4 15lx10~ pfu 300 mg/kg ~y 0 AvllacZ4 16lx10~ pfu 300 mg/kg ~y 0 AvllacZ4 17 none 5 mg/kg Dex 0 18 none .5 mg/kg Dex 0 19lx108 pfu 5 mg/kg Dex 8 AvllacZ4 20lx108 pfu 5 mg/kg Dex 8 AvllacZ4 One week after administration of AvlH9F2, plasma was prepared and analyzed by ELISA to determine ~he levels of hl-m~n Factor IX. The results are shown in Figure 17. Mice which received AvllacZ4 without immunosuppression, then received AvlH9F2 five weeks later, expressed no human Factor IX. Mice which received neither immunosuppression nor AvllacZ4, but were treated with AvlH9F~, expressed an average of 9.2 ~g/ml. Mice which were immunosuppressed with DSG at the time of AvllacZ4 deiive~y, and the- rece'ved AvlH9F2, expressed an average of 6.6 ~g/ml. ~ c- which were immunosuppressed with DSG, but received ~.o AvllacZ4, and then treated with. AvlH9F2, had an average level of 5.2 ~g/ml.
Finally, mice which were treated wi~h DSG, but received neither vector, expressed no human Facto~

Mice immunosuppressed with a ~ow dose of cyclophosphamide at the time of AvllacZ~ admln stration, did not express human Factor IX after delive~y of AilH9F2. Mice treated with a high dose of cyclophosphamide, but not administered AvllacZ4, expressed an average of 8 ~g/ml one 2200~69 WO96/12406 PCT~S95/13253 week after delivery of AvlH9F2. Mice immunosuppressed with a high dose of cyclophosphamide at the time of AvllacZ4 treatment, expres-~ed an average of 15.4 ~g/ml of human Factor IX after treatment with AvlH9F2. Mice treated with cyclophosphamide, but not treated with either adenoviral vector, did not express human Factor IX.

Mice immunosuppressed with dexamethasone, bu. not treated with AvllacZ4, expressed an average of 5.5 ~g/ml of hl~m~n Factor IX one week after administration of AvlH9F2.
However, mice which received AvllacZ4 at the time of immunosuppression did not express Factor IX after delivery of AvlH9F2. Mice which were immunosuppressed, but not treated with either vector, did not express human Factor IX.

ExamPle 4 Su~Dresslon of humoral immune resDonse to adenoviral vectors to enable the re~eat administrations thereof This example is an ela~oration and expansion of the data contained in Example 3. In this example, the following materials and methods were employed.

Adenoviral vectors A~;LacZ4 and AvlH9F2 were aescribed in Example 3 nereinabove. AvlH9FR was made by cotransfecting 293 cells with pAvlH9FR (Figure l5) with tAe large DNA fragment from ClaI d1gested Ad dl327. Recombinan~ adenoviral vector plaques were picked, expanded, and screened for expression of Factor IX by ELISA. A positive clone was identified and amplified, thus generating the vector AvlH3FR. This vector, like AvlH9F2, contains a centrally truncated first intron and the compleee 5' and 3' untranslated regions from the human Factor IX gene. The centrally truncated first intron and 3' untranslated region are essentially the same sequences 22~0869 WO96112406 PCT~S95/132~3 described by Jallat, et al., EMBO J., Vol. 9, pgs. 3295-3301 ( 1990 ) .

AvlALAPH81 is an adenoviral vector which contains the B-domain deleted human Factor YIII cDNA expressed from the mouse albumin promoter, and is described in published PCT
Application No. WO94/29471.

All vector stocks contained less than 1 in 106 wild-type adenovirus, as determined by quantitative PCR analysis of Ela sequences.

Im}nunosuppressants Deoxyspergualin (manufactured by Nippon Rayaku Co., Ltd., Tokyo, Japan) was a gift from Bristol-Myers-Squibb, Princeton, N.J. A 100 mg vial of deoxyspergualin was reconstituted with water to a final concentration of 25 mg/ml, aliquoted, and frozen at -70C. Frozen stocks were thawed at room temperature and diluted with Hanks Balanced Salt Solu~ion (BSS) prior to in~ectior..

Deoxyspergualin is an immunosuppressant currently being tested clinically in organ transplantation. It has a potent, long ter~ effect on antigen specific B cells and has been showr to prevent effectively the production of specific antibody when co-administered with protein antigens.
(Alegre, et al ., TransPlantation, Vol. 57, pgs. 1786-1794 (1994'; Tepper, Ann. N. Y. Acad. Sci., pgs. 123-132 (1993);
Tufveson, et al., TransPlant. Proc., Vol. 26, pgs. 3029-3039 (1994)j. The mode of action cf DS5 is not fully understood at the molecular level. The data suggest that it may interfere with differentiation of t3 and T cells and also with antigen processing. Recent studies showed that DSG inhibited K light chain expression and therefore blocked IgM expression on the surface of pre B cells. (Tepper, 1993). In addition, 22008~9 WO96/12406 PCT~S95/13253 the data showed that DSG inhibited nuclear translocation of NFKB (Tepper, 1993), which could be the mechanism by which DSG inhibits differentiation of B and T cells. Finally, it has been ~o~Rtrated that DSG binds to Hsc70, a heat shock protein. (Tepper, 1993). Heat shock proteins are involved in protein folding, molecular chaperoning, peptide loading of MHC molecules, and antigen presentation. Therefore, binding of DSG to Hsc70 may explain the effect on antigen presentation.

Cyclophosphamide (Cy) was obtained from Sigma and Dissolved in HBSS. Dexamethasone (Dex) solution from American Regent Laboratories, Inc., Shirley, N. ~-. was diluted in HBSS prior to injection. All three immunosuppressants were delivered intraperitoneally, according to the doses indicated hereinbelow.

~n; m~ 1 procedures C57BL/6 mice were obtained from Harlan Sprague Dawley (Indianapolis, IN). Adenoviral vectors were administered via tail vein injection after diluting the appropriate amount of vector stock to 0.5 ml with Hanks Balanced Salt Solution (HBSS). At the time points indicated in the text, blood was obtained from the retroorbital plexus. For preparation of plasma samples, sodium citrate was added immediately to a final concentration of 0.38~ (w/v). To prepare sera samples, the biood was allowed to clot. Samples were centrifuged for 5 min. in an Eppendorf Microfuge after which the plasma or serum was collected, aliquoted, and frozen.

Human Factor VIII ELISA
Plasma levels of human Factor VIII were determined by ELISA, as described in Connelly, et al., Human Gene Therapy, Vol. 6, pgs. 185-193 (1995). The limit of sensitivity with mouse plasma samples containing B domain deleted human Factor SUBSTITUTE SHEET (RULE 26) 220086~

WO96/12406 PCT~S95/13253 VIII was 3 to 6 ng/ml. Mouse plasma samples were diluted 1:5 prior to the assay, therefore, the actual limit of detection was 15 to 30 ng/ml.

Human Factor IX ELISA
Plasma levels of human Factor IX were determined by ELISA. Asserachrom IX:Ag ELISA kits were purchased from American Bioproducts Company (Parsippany, NJ) and assa~-s were performed according to the manufacturer's instructions. The limit of sensitivity was 1.6 ng/ml.

Anti-adenoviral antibody assay Mouse plasma or serum samples were heat inactivated at 55C for 30 minutes and then diluted in Improved Minimal Essential Medium (Biofluids, Rockville, MD) plus 2~ FBS
(IMEM/2~FBS) in two-fold steps beginning at 1:2. 55 ~1 of each sample were mixed with 10 ~1 of AvllacZ4 (containing 4 x 10' pfu), incubated for 1 hour at 37C and applied to nearly confluent 293 cells in 96 well plates (4 x lG~ cells per well). After 50 minutes in the tissue culture incubator, the virus was aspirated from each well and re~laced with 150 ~1 of IMEM/lO~FBS. The following day, cells were fixed and stained for ~-galactosidase expression, as described previously. (Smith, e~ al., Nature Genetics, Vol. 5, pgs.
397-402 (1993?). In the absence of inactivating antibodies, all of the cells stained blue. The titer of inactivating antibodies for each sample was reported as the reciprocal of the highest dilution with which less than 25~ of the cells stained blue.

Results Dose dependence of the humorai im~une rçsPonse to adenovirus vectors Previous studies which have ~ml ned repeat delivery of adeno~irus vectors have employed relatively high doses of WO96112406 PCT~S95113253 vector, which would be expected to maximize the strength of the immune response. (Smith, et al ., 1993; Kozarsky, et al ., J. Biol. Chem., Vol. 269, pgs. 13695-13702 (1994); Kay, et al., Proc. Nat. Acad. Sci., Vol. 91, pgs. 2353-233357 (1994~;
Yei, et al., Gene TheraPy, Vol. 1, pgs. 192-200 (1994); Yang, et al., J. Virol., Vol. 69, pgs. 2004-2015 (1995); Dai, et ai., Proc. Nat. Acad. Sci., Voi. 92, pgs. 14ûi-14û5 [i995~;
Barr, et al., Gene TheraPY, Vol. 2, pgs. 151-155 (1995)).

To determine whether the production of neutralizing antibodies and the block to repeat delivery is dependent on the dose of vector inoculated, various doses of the adenovirus vector AvllacZ4 to C57BL/6 mice via tail vein were administered. The vector inoculum ranged from 1 x 103 pfu to 1 x lû~ pfu in single log increments. Thirty-four days after vector delivery, the serum levels of anti-adenovirus neutral i zing antibodies were determined (~ig. 18) for mice which received lx10' pfu or greater of vector. A minus sign indicates that none of the mice in the cohort had detectable antibody. The plus sign corresponding to mice which received lxl o3 pfu of vector indicates that three of tne five mice had an antibody titer of 8, while two mice had r.o detectable antibody. Thus, three of five mice which received 1 x 108pfu had a level of anti-adenovirus antibody wnich was sufficient to neutralize 4xlC` pfu of AvlLacZ4, whiie two mice had undetectable levels. None of the mice which received lower doses of vector had detectable antibodies using this relatively stringent neutralization assa-y.

Thirty-five days after administraticn of AvllacZ4, each mouse received 2 x 10~ pfu of AvlH9FR, an adenoviral vector encoding human Factor IX. One week later, the plasma levels of human Factor IX were determined by ELISA (Fig. 18). An average of approximately 2 ~g/ml of Factor IX was detected in mice which received either no AvllacZ4, or up to 1 x 105 pfu WO96/12406 2 2 0 0 8 6 9 PCT~S95/13253 ~

of the first vector. Factor IX was also readily detected in the mice which had received a first dose of l x 106 and l x 107 pfu, although the levels were reduced. Mice which received l x 108 pfu of AvllacZ4 yielded little or no human Factor IX after a~m; n; ~tration of AvlH9FR. Thus effective gene transfer and expression can be achieved with a second vector ~m; n; stration, provided the initial vector dose is below a certain threshold level. The data indicate that for intravenous delivery in C57BL/6 mice, this value is between 107 and 108 pfu.

Transient immunosuppression increases the efficiency of vector re-administration. ~-To determine whether transient immunosuppression wouldallow readministration of an adenoviral vector, C57BL/6 mice were immunosuppressed with either deoxyspergualin (DSG), cyclophosphamide (Cy), or ~ methasone (Dex) at the time of administration of l x 108 pfu of AvliacZ4. As shown above, this dose completely prevented an effective second delivery.
Mice were injected daily with 33 mg/kg of DSG, beginning one day before vector delivery and continuing for seven more days. Dexamethasone was delivered over the same time course, at a dose of 5 mg/kg. Cyclophosphamide was administered once, the day before vector delivery, at a dose of either lO0 mg/kg or 300 mg/kg. Control mice received AvllacZ4 without immunosuppression, or were immunosuppressed without initial vector delivery.

Five weeks after vector ~m; n; stration, the plasma levels of anti-adenovirus neutralizing antibodies were determined (Fig. l9). Mice immunosuppressed with DSG or 300 mg/kg of cyclophosphamide had no detectable neutralizing antibodies, but all other mice which received AvllacZ4 generated neutralizing antibodies. Thirty-five days after the first vector in~ection, the mice received l x 108 pfu of SU85T~IlJTE SHEET(RULE 26) ~ WO96/12406 2 2 0 0 8 6 ~ PCT~S9~/13253 AvlH9F2. One week after AvlH9F2 injection, plasma samples were prepared and the levels of human Factor IX were determined by ELISA (Fig. 17). Mice immunosuppressed with DSG or 300 mg/kg cyclophosphamide expressed human Factor IX
at levels which were approximately the same as levels in mice which had not been treated with the first vector. No human Factor IX was detected in the plasma of the mice which were not immunosuppressed at the time of AvllacZ4 administration.
Under the conditions used in these studies, immunosuppression with dexamethasone or l00 mg/kg cyclophosphamide was not effective in permitting expression of Factor IX on repeat injection. Thus the ability to achieve transgene expression on a second vector injection correlated with the suppression of neutralizing antibodies by DSG or high dose cyclophosphamide treatment.

At the time of administration of the second vector, AvlH9F2, half of the mice in each cohort which had been immunosuppressed with cyclophosphamide or dexamethasone were immunosuppressed again, using the same regimen as at the first vector delivery. Five weeks later, plasma levels of anti-adenovirus neutralizing antibodies were determined. The mice immunosuppressed with 300 mg/kg cyclophosphamide had no detectable neutralizing antibodies, while mice im~.unosuppressed with l~. mg/kg cyclophosphamide or 5 mg/kg dexamethasone had a measl~rable response (data not shown).
Thir~y-five days after AvlH9F~ injection, l x 109 pfu of a Factor VIII vector, AvlAllPH81, were administered to the mice which had been immunosuppressed with 300 mg/kg cyclophosphamide. In addition, Factor VIII vector was administered to control mice which had received AvllacZ4 and AvlH9F2 without immunosuppression and also to naive mice.
One week later, plasma levels of human Factor VIII were determined by ELISA ~Fig. 20). Control mice which received only the AvlALAPH81 vector, and mice immunosuppressed with 22008 6q Wo96/12406 PCT~S95/13253 300 mg/kg of cyclophosphamide at the time of the two prior vector injections expressed human Factor VIII. Mice which received AvllacZ4 and AvlH9F2 without imunosuppression, as well as mice which were immunosuppressed only at the time of AvllacZ4 delivery, did not express human Factor VIII.

DSG ~ermits effective rePeat administration at a clinically relevant dose.
The previous experiment demonstrated that a high dose of either DSG or cyclophosphamide permitted readministration of an adenoviral vector, but that clinically relevant doses of cyclophosphamide and dexamethasone were not effective. The next objective was to determine whether lower doses of DSG
would be effective. The dose of DSG used in the initial experiment (33 mg/kg) is near the maximum tolerated dose in mice and is significantly higher than the 5-7 mg/kg dose used in human clinical trials for organ transplantion. (Suzuki, et al., Ann. N.Y. Acad. Sci., Vol. 696, pgs. 263-269 (1993);
Jindal, et al., Mt. Sinai J. Med., Voi. 61, pgs. 51-56 (1994~). To determine if lower doses would also be effective in allowing vector readministration, mice were immunosuppressed with 5, 10, 20, and 33 mg/kg of DSG at the time of administration of ' x 10~ pfu of AvllacZ4.
Immunosuppression was started the day before vector deli~ery and continued for a total of 8 days. Or. the day of vector d~livery, DSG was given after injection of the adeno~irus since it is most effective when administered after antigen.
(Takahara, et al., TransPlantation, Vol. 53, pgs. 514-918 (lg92) ) .

Thirty-five days later each mouse received 1 x 10~ pfu of AvlH9F2. One week after AvlH9F2 injection, human Factor IX plasma levels were determined by ELISA (Fig. 21j. Control mice, which were not pre-immunized with AvlLacZ~, expressed an average of 9 ~g/ml of human Factor IX. Other control ~200869 ~ WO~6/12406 PCT~S95tl3253 `_ mice, which received AvllacZ4 but were not immunosuppressed, expressed no hllm~n Factor IX after AvlH9F2 a~m; n; ~tration.
The one mouse which was ;mmllnosuppressed with 33 mg/kg DSG
- expressed 12 ~g/ml of human ~actor IX. Five of six mice ;mmllnosuppressed with 20 mg/kg DSG expressed an average of 3.0 ~g/ml of hllm~n Factor IX, and one mouse expressed none.
~ice immunosuppressed with a dose of 10 mg/kg expressed a wide range of Factor IX, extending from 30 ng/ml to 8 ~g/ml.
Three mice immunosuppressed with 5 mg/kg expressed no human Factor IX, while three others expressed levels ranging from 2 to 7 ~g/ml. Mice which were not immunosuppressed at the time of AvllacZ4 administration expressed no hl7m~n Factor IX.

Discussion The data ~emo~trate that multiple intravenous administrations of adenovirus vectors with resulting transgene expression can be accomplished in immune competent animals treated with a short course of immunosuppression at the time of vector delivery. This observation is significant because several recent studies have demonstrated that a humoral immune response directed against adenoviral vector prevents readministration. (Smith, et al., 1993; Kay, et al., 1994; Yei, et al., 1994; Yang, et al., 1995.) The inability to readminister vector has presented a major obstacle to the clinical utility of adenoviral vectors, since effective readministration will almost certainly be required for the clinical application of these vectors to gene therapy of chronic diseases.

The failure to obtain expression following repeat dosing in the absence of immunosuppression correlates with anti-adenovirus neutralizing antibodies. Evidence that such antibodies are sufficient to block readministration was provided by Yang et al., 1995, who showed that passive transfer of serum from a mouse previously treated with vector SUBSTlTlJTE SHEET (RULE 26) 22oo86q PCT~S95/13253 into the venous circulation of a naive mouse was able to prevent vector-mediated gene expression in the liver. The role of the immune system in preventing repeat administration also was demonstrated by the observation that repeat administration of vector was effective in immunodeficient mice. (Yang, et al ., 1995; Dai, et al ., 1995; Barr, et al ., Gene TheraPy, Vol. 2, pgs. 151-155 (1995)). This result has been confirmed by demonstrating effective administration of AvlH9F2 to scid mice 5 weeks after delivery of 5 x 108 pfu of AvlALAPH81 (data not shown).

Since previous reports describing humoral responses to adenovirus administration used relatively high vector doses, we evaluated the relationship between initial vector dose and the ability to achieve an effective repeat gene transfer.
The results indicated that the magnitude of the immune response was dependent on the initial dose of vector and that if the dose is below a threshold level, a second a~m;n;stration is possible. The finding that this level was only one to two orders of magnitude below a clinically relevant vector dose further suggested that immunosuppression at the time of vector delivery would permit readministration.
In addition, these results emphasize the need for potent vectors which would be efficacious at low doses.

Applicants have observed that an~i-adenovlrus neutralizing antibody titers are maintained for at least ten monthc in mice after a single administration of vector via tail vein. The long-term maintenance of titer may have been due to a low level of ongoing adeno~iral backbone gene expression n ~,ansduced cells. Vectors designed to red~ce or eiiminate backbone gene expression may eiicit a weaker immune response and therefore may require less immunosuppression for successfui readministration.

220086q WO96/12406 PCT~S95/132~3 An important property of DSG is that it does not produce a general suppression of the immune system, but rather results in a selective lack of response to specific antigens presented at the time of drug treatment. We found that immunosuppression with DSG over a 7 day period following vector delivery efficiently inhibited the humoral response to the vector and permitted an effective second administration.
The initial experiment with DSG employed a high dose of 33 mg/kg, which is close to the m~ m tolerated dose in mice and several fold higher than the doses used in human trials.
When administered over the same 8 day course, including 7 days post vector treatment, lower doses of DSG were also effective in permitting repeat delivery of vector. A greater degree of individual variability in levels of Factor IX
expression was seen with reduced doses, although even at the lowest dose tested (5 mg/kg) significant Factor IX expression was obtained in 3 of 6 animals.

~ yclophosphamide, administered at a dose of 300 mg/kg the day before vector in~ection, was also effective in blocking the humoral response and allowed a completely effective second injection with a Factor IX adenovirus vector Furthermore, a third injection with a Factor VIII
encoding vector was also completely efficacious when the previous two vector administralions we~e each preceded by a singie dose of cyclophosphamide. ~yclophosphamide is used clinicaliy as an anti-cancer agent in the treatment of Hodgkins disease and otner leukemias. It is also empioyed as an i~unosuppressive agent in the treatmer- of hemophilia patients who develop inhibitors to Factor VIII protein replacement therapy. (Aledort, Am. J. Xemat., Vol. 47, pgs.
208-2,7 (1994); Nilsson, et al ., N. Enql. ~. Med., Vol. 318, pgs. 947-950 (1988)i. W~ile the dose used t C successfully obtain readministration in mice is substantially higher than is generally used in humans, it remains tO be established WO96/12406 2 2 0 0 8 6 ~ PCT~S95/13253 ~
whether lower, clinically acceptable doses, might be effective in humans. One possibility suggested by experience in the organ transplantation setting is that combinations of immunosuppressants would yield more potent suppression of the immune system with less toxicity. For example, cyclophosphamide may be effective at lower doses when used in combination with dexamethasone. It is also possible that the degree of immunosuppression required will depend on the dose of vector which is needed to effect therapy. The dose of AvlH9F2 used in this study, l x l0~ pfu, yielded plasma levels of human Factor IX of 5-l0 ~g/ml, which i8 20 to 50 times above a level that would be therapeutic in a hemophiliac. Vectors, such as AvlH9F2, which express high levels of transgene product and which can be administered at relatively low doses, should reduce the extent of immune stimulation and the degree of immunosuppression required.

In summary, Applicants have shown that effective repetitive delivery of systemically administered adenovirus vectors can be a_hieved with short term immunosuppression.
Importantly, this can be accomplished using pharmacologic agents wnich are either approved for use in humans, or are in clinical testing.

The disclosure of all patents, publicarions, (including published patent applicationsi, and database accession numbers, and deposi~ory accession numbers referenced in this specifica.ion are speci~ically incorporated herein by reference in their entirety to the same extent as if each such individual patent, publication, and d~tabase accession number, and depository accession number were specifically and individual~y indicated .c be ncorporate~ by reference.
.
It is to be understood, ;nowever, that the scope of the present invention is no. to be limited to the specific 22008~9 096/12406 PCT~S95/132~3 embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims.

Claims (21)

WHAT IS CLAIMED IS:

1. A method of effecting a gene therapy treatment in a host, comprising the steps of:
(a) administering to a host (i) an adenoviral vector including at least one DNA sequence encoding a therapeutic agent and (ii) an immunosuppressive agent;
(b) discontinuing said administration of said adenoviral vector and said immunosuppressive agent; and (c) repeating the course of administration of said adenoviral vector including at least one DNA sequence encoding a therapeutic agent, and said immunosuppressive agent of step (a) at least once, said adenoviral vector being administered in an amount effective to produce a therapeutic effect in said host, and said immunosuppressive agent being administered in an amount effective to prevent or suppress an immune response against said adenoviral vector in said host.

2. The method of Claim 1 wherein said immunosuppressive agent is a steroid.

3. The method of Claim 2 wherein said steroid is dexamethasone.

4. The method of Claim 1 wherein said immunosuppressive agent is cyclosporin A.

5. The method of claim 1 wherein said adenoviral vector is administered each time in an amount of from about 1 pfu to about 10 14 pfu.

6. The method of Claim 3 wherein said dexamethasone is administered each time in an amount of from about 1 mg/kg to about 15 mg/kg.

7. The method of Claim 6 wherein said dexamethasone is administered each time in an amount of about 2 mg/kg.

8. The method of Claim 5 wherein said adenoviral vector is administered each time in an amount of from about 10 6 pfu to about 10 13 pfu.

9. The method of Claim 8 wherein said adenoviral vector is administered each time in an amount of from about 10 8 pfu to about 10 10 pfu.

10. The method of Claim 1 wherein said immunosuppressive agent is deoxyspergualin.

11. The method of Claim 10 wherein said deoxyspergualin is administered each time in an amount of from about 1 mg/kg to about 33 mg/kg.

12. The method of Claim 1 wherein said immunosuppressive agent is cyclophosphamide.

13. The method of Claim 12 wherein said cyclophosphamide is administered each time in an amount of from about 5 mg/kg to about 300 mg/kg.

14. The method of Claim 1 wherein said deoxyspergualin is administered each time in an amount of from about 3 mg/kg to about 7 mg/kg.

15. The method of Claim 13 wherein said cyclophosphamide is administered each time in an amount of from about 50 mg/kg to about 100 mg/kg.

16. The method of Claim 1 wherein said immunosuppressive agent is administered for a period of time which does not exceed 14 days.

17. The method of Claim 16 wherein said immunosuppressive agent is administered for a period of time which does not exceed 11 days.

18. The method of Claim 17 wherein said immunosuppressive agent is administered for a period of time which does not exceed 8 days.

19. The method of Claim 1 wherein said administration of said immunosuppressive agent is begun at about 24 hours prior to administration of said adenoviral vector.

20. The method of Claim 1 wherein said administration of said immunosuppressive agent is begun at the same time as administration of said adenoviral vector.

21. The method of Claim 1 wherein said administration of said immunosuppressive agent is begun at about 24 hours after administration of said adenoviral vector.