US006093699A United States Patent (19) 11 Patent Number: 6,093,699 Sehon et al. (45) Date of Patent: Jul. 25, 2000

54 METHOD FOR GENE THERAPY Sehon, A.H., "Conversion of Antigens to Tolerogenic INVOLVING SUPPRESSION OF AN IMMUNE Derivatives by Conjugation w/mPEG” (1987) Molecular RESPONSE Cell Biology, 65:pp. 205-219. 75 Inventors: Alec Sehon, Winnipeg, Canada; Judith Marshall, Eliot, “Gene Therapy’s Growing Pains” (1995), A. Kapp, Atlanta, Ga., Glen M. Lang, Science, 269: pp. 1050–1054. Winnipeg, Canada; Yong Ke, Carmel, “Report and Recommendations of the Panel to Assess the Ind. NIH Investment in Research on Gene Therapy” (1995). Sehon, S.H., “Down Regulation of IgE Antibodies by Sup 73 Assignees: The University of Manitoba, pressogenic Conjugates' (1983) Academi Press Japan, Inc. Manitoba, Canada; Emory University, pp. 483-491. Atlanta, Ga. Sehon, A.H., “Modulation of Antibody Responses by Con 21 Appl. No.: 08/339,245 jugates of Antigens with Monomethoxypolyethylene Gly col”, from Immunobiology of Proteins and Peptides V, 22 Filed: Nov. 10, 1994 Plenum Publishing Corp., (1989). Related U.S. Application Data Sehon, A.H., “Immunological Strategies for Therapeutic Destruction and HIB-Infected Cells in Asymptomatic 63 Continuation-in-part of application No. 07/877,368, May 4, Patients”, MCR Group for Allergy Research, pp. 570-574. 1992, Pat. No. 5,447.722, which is a continuation of appli cation No. 07/707,972, May 23, 1991, abandoned, which is Sehon, A.H., Potential use of Allergens Modified with a continuation of application No. 07/478,049, Feb. 7, 1990, Monomethoxypolyethylene Glycol for Immunotherapy of abandoned, which is a continuation of application No. Atopic Patients, pp. 313–318. 07/071.462, Jul. 9, 1987, abandoned. Handbook of Experimental Immunology, 2nd edition, Ed. 51 Int. Cl." ...... A01N 43/04; A61K 31/70; S.M. Weir, Blackwell Scientific Publication, Oxford 1973, p. C12N 15/00; C12N 15/63 35.3. 52 U.S. Cl...... 514744; 435/320.1; 435/455; 424/93.21; 424/178.1; 424/193.1; 424/280.1; Bitoh et al. (1993) Cellular Immunol 150: (68-93. 424/810; 424/85; 530/389; 530/391 Culver M.D., Kenneth W., “Gene Therapy”, A Handbook for 58 Field of Search ...... 514/44; 424/178.1, Physicians. 424/193.1, 280.1, 810, 85, 93.21; 530/389, 391; 435/320.1, 455 Primary Examiner Deborah Crouch ASSistant Examiner Jill D. Martin 56) References Cited Attorney, Agent, or Firm- Antonelli, Terry, Stout & Kraus, U.S. PATENT DOCUMENTS LLP 4,261,973 4/1981 Lee et al...... 424/78.3 57 ABSTRACT 4,276,206 6/1981 Katz ...... 525/54.1 4,296,097 10/1981 Lee et al. .. 424/78.28 A method for Specifically Suppressing the capacity of a 4,430.260 2/1984 Lee et al...... 525/61 mammal receiving gene therapy to mount an immune 4,650,675 3/1987 Borel ...... 424/85 response to a given expressed gene of the deficient gene in 5,358,710 10/1994 Sehon ...... 424/178.1 question, which response is caused by the administration of 5,399,346 3/1995 Anderson ...... 424/93.21 “foreign' DNA encoding an antigenic protein or vectors OTHER PUBLICATIONS comprising Such DNA for expression of the deficient gene in Sehon, A.H., “Cytokine Gene Expression of CD8+ Suppres the patient receiving gene therapy. Sor T Cells Induced by Tolerogenic Conjugates of Antigen and mPEG” (1993) Cellular Immunology, 149: pp. 409–421. 3 Claims, 2 Drawing Sheets U.S. Patent Jul. 25, 2000 Sheet 1 of 2 6,093,699

FIGURE 1

16384 8 192 4O96 CONTROL : PBS 2O48 1 O24 (59%) 512 256 128 (94%) 64 32 16 (99%) 8 O 4 (100%) (100%) -- T -t-t-t-t-ar---- 1 2 3 4 5 6 7 8 9 10 1 1 12 mPEG GROUPS per OA

FIGURE 2

1 6384 8, 192 4096 Control : PBS 2O48 1024 512 256 128 64 32

18 A

O (e.9A9588.75. -(O)

1O 30 5O 70 90 1 10 13O 15O DOSE OF OA-m PEG (Aug protein) U.S. Patent Jul. 25, 2000 Sheet 2 of 2 6,093,699

FIGURE 3

64OO 32OO CONTROL : PBS 16OO 8OO 4OO (86%) 2OO O (91%) (86%) (90%) 1OO O (93%)

1 2 3 4 5 6 7 8 9 10 1 1 12 mPEG GROUPS per OA 6,093,699 1 2 METHOD FOR GENE THERAPY appropriate gene may Vitiate the desired therapeutic effects. INVOLVING SUPPRESSION OF AN IMMUNE This invention provides a method for overcoming this RESPONSE inherent complication due to the immunogenic capacity of the expressed protein, and is therefore considered to repre This application is a continuation-in-part of pending U.S. 5 Sent a novel and an essential improvement for the treatment application Ser. No. 07/877,368, filed on May 4, 1992 by of Such diseases. Lang et al., now U.S. Pat. No. 5,447,722, which is a AS Background to the present invention: continuation of abandoned U.S. application Ser. No. 07/707, Chen, Y., Takata, M., Maiti, P. K., Mohapatra, S., 972, filed on May 23, 1991 by Lang et al., which is a Mohapatra, S. S. and Sehon, A. H., disclose that the Sup continuation of abandoned U.S. application Ser. No. 07/478, preSSor factor of TS cells induced by tolerogenic conjugates 049, filed on Feb. 7, 1990 by Lang et al., which is a of OVA and mPEG is serologically and physicochemically continuation to abandoned U.S. application Ser. No. 07/071, related to the CfB heterodimer of the TCR. J. Immunol. 462, filed on Jul 9, 1987 by Lang et al. 152:3-11, 1994. FIELD OF THE INVENTION Mohapatra, S., Chen, Y., Takata, M., Mohapatra, S.S. and 15 Sehon, A. H. disclose “Analysis of TCR Of chains of CD8" The present invention relates to a method for Suppressing Suppressor T cells induced by tolerogenic conjugates of the capacity of a mammal to mount an immune response antigen and monomethoxypolyethylene glycol: Involvement caused by the administration of one or more immunogenic of TCR C-CDR3 domain in immuno-suppression.” J. Immu therapeutic material(s), Such as gene vectors or their expres nol. 151:668–698, 1993. Sion proteins used in applications to gene therapy. Bitoh, S., Takata, M., Maiti, P. K., Holford-Stevens, V., BACKGROUND Kierek-Jaszczuk, D. and Sehon, A. H., disclose that "Antigen-specific Suppressor factors of noncytotoxic CD8" Foreign proteins or DNA, Such as genetic material or Suppressor T cells downregulate antibody responses also to vectors for gene therapy, or their derivatives, have thera 25 unrelated antigens when the latter are presented as peutic properties and are administered to patients Suffering covalently linked adducts with the Specific antigen.” Cell. from certain diseases. However, as discussed later, the Immunol. 150:168-193, 1993. immunogenicity of the Said foreign proteins, nucleotides, Bitoh, S., Lang, G. M., Kierek-Jaszczuk, D., Fujimoto, S. DNA or vectors, or of their derivatives, may vitiate the and Sehon, A. H., disclose “Specific immunosuppression of treatment and hence this invention provides an improved human anti-murine antibody (HAMA) responses in hu-PBL method for the treatment of Such diseases. SCID mice.” Hum. Antibod. Hybridomas 4:144-151, 1993. Gene therapy is the insertion of a functioning gene into Bitoh, S., Lang, G. M. and Sehon, A. H., disclose the the cells of a patient (i) to correct an inborn error of “Suppression of human anti-mouse idiotypic antibody metabolism (i.e., genetic abnormality or birth defect result responses in hu-PBL-SCID mice.” Hum. Antibod. Hybrido ing in the deficiency of the patient with respect to one or 35 more essential proteins Such as enzymes or hormones), or mas 4:144-151, 1993. (ii) to provide a new function in a cell (Kulver, K.W., “Gene Dreborg, S. and Akerblom, E., disclose the Safety in Therapy”, 1994, p. xii, Mary Ann Liebert, Inc., Publishers, humans of “Immunotherapy with monomethoxypolyethyl New York, N.Y.). ene glycol modified allergens.” In: S. D. Bruck (Ed.), CRC Crit. Rev. Ther. Drug Carrier Syst. 6:315-363, (1990). When the host is totally deficient of the inserted gene from 40 birth, the new protein expressed by this gene-when the Generally the term antigen refers to a Substance capable latter is inserted into the appropriate cell of an adult host of eliciting an immune response and ordinarily this is also would be expected to induce in the host an immune response the Substance used for detection of the corresponding anti against itself. Hence, (i) the host would produce antibodies bodies by one of the many in vitro and in vivo immuno or cytotoxic cells to the “new” protein, and (ii) this immune 45 logical procedures available for the demonstration of response would not only combine and neutralize and thus antigen-antibody interactions. inactivate the function of the “new” protein, but may also Similarly, the term allergen is used to denote an antigen lead to untoward therapeutic complications due to formation having the capacity to induce and combine with reaginic of immune complexes. It is, therefore, not Surprising that (i.e., IgE) antibodies which are responsible for common gene therapy has proven Successful in adenosine deaminase 50 allergies; however, this latter definition does not exclude the (ADA) deficiency, i.e., in children deficient of ADA from possibility that allergens may also induce reaginic birth, which is manifested by the absence of functional T antibodies, which may include immunoglobulins of classes lymphocytes and consequently to the Severe combined other than IgE. immunodeficiency (SCID) syndrome. The reported success AS used herein, the term antigenicity is defined as the of gene therapy in young children deficient of ADA from 55 ability of an antigen (immunogenic material) or allergen to birth is related to the immunodeficient status of the child, as combine in Vivo and in vitro with the corresponding anti no immune response can be generated against the foreign bodies, the term allergenicity or Skin activity is defined as therapeutic genetic material. As a corollary, gene therapy the ability of an allergen to combine in vivo with homolo would be Successful it it is instituted from birth, when it is gous reaginic antibodies thereby triggering Systemic ana relatively easy to induce immunological tolerance to a 60 phylaxis or local skin reactions, the latter reactions being the foreign immunogenic material. result of direct skin tests or of passive cutaneous anaphy Foreign immunogenic materials, Such as biologic lactic (PCA) reactions; and the term immunogenicity in a response modifiers or their derivatives, often have therapeu general Sense is the capacity of an antigen or allergen, or of tic properties and are, therefore, administered to patients their derivatives produced in vitro or processed in Vivo, to Suffering from certain diseases. However, as a result of the 65 induce the corresponding Specific antibody response. immunogenicity of the foreign materials, or of their In relation to this invention, tolerogens are defined as derivatives, for the reasons stated above the insertion of the immunosuppressive covalent conjugates consisting of an 6,093,699 3 4 antigenic material (immunogenic proteins, etc.) and a water jugate comprising the genetic material and/or its expression Soluble polymer (see e.g. Sehon, A. H., In “Progress in product (i.e., the protein of which the patient is deficient) Allergy” (K. Ishizaka, ed.) Vol. 32 (1982) pp. 161-202, and monomethoxypolyethylene glycol having a molecular Karger, Basel; and U.S. Pat. No. 4,261,973). weight of about 4,500-10,000 daltons, the above adminis In the present context and claims the term tolerogen thus tration being at least one day prior to administration of the refers to a conjugate consisting of an immunogenic material therapeutic genetic material for gene therapy, wherein Said (protein or polynucleotide) and a nonimmunogenic method results in the Specific Suppression of the immune conjugate, Said tolerogen being immunosuppressive in an response and the active development of Specific tolerance to immunologically Specific manner with respect to the antigen Said therapeutic genetic material and/or its expression which is incorporated into the tolerogenic conjugate irre product(s). Spective of the immunoglobulin class which is downregu In a preferred embodiment the therapeutic genetic mate lated; furthermore, the tolerogen may comprise a conjugate rial is selected from nucleotides, DNA, RNA, MRNA, of an essentially nonimmunogenic polymer and an immu attached to appropriate vectors for expression of the required nogenic biologically active product or derivative of the therapeutic protein. genetic material used for gene therapy. 15 In a more preferred embodiment gene therapy vectors The therapeutic administration of foreign immunogenic include Moloney murine leukemia virus vectors, adenovirus material induces an immune response leading to the forma vectors with tissue Specific promoters, herpes simplex tion of antibodies of different immunoglobulin classes. vectors, vaccinia Vectors, artificial chromosomes, receptor Hence, on repeated administration, the material may form mediated gene delivery vectors, and mixtures of the above complexes in Vivo with Such antibodies leading to a poor VectOrS. therapeutic effect by Virtue of its being Sequestered and neutralized by the antibodies, or to anaphylactic reactions by BRIEF DESCRIPTION OF THE DRAWINGS combination with reaginic antibodies, or to other untoward conditions, i.e. immune complex diseases due to the depo FIG. 1 is a diagram illustrating the Suppressogenic rela 25 tionship between treatment of (B6D2)F1 mice with Sition of antibody-antigen complexes in Vital tissues and OA-mPEG conjugates of different molecular compositions Organs. and the primary anti-OAIgG response, as measured by PCA Wilkinson et al. “Tolerogenic polyethylene glycol deriva titers. The percentages in brackets represent the degree of tives of Xenogenic monoclonal immunoglobulins', Immu Suppression with respect to the minimal immune response in nology Letters, Vol. 15 (1987) pp. 17-22, discloses the animals receiving phosphate buffered saline (PBS) in lieu of criticality of the administration time of a tolerogenic con the conjugates. jugate to a non-Sensitized individual at least one day prior to FIG. 2 is a diagram illustrating the Suppressogenic rela challenge with an antigen. tionship between the treatment of (B6D2)F1 mice with The present invention overcomes deficiencies of the prior different dosages of OA-mPEG conjugates of different art, providing a means for inducing a priori tolerance to a 35 molecular compositions and the primary anti-OA IgG protein or polynucleotide in an individual deficient of the given protein or polynucleotide, thus making the adminis response, as measured by PCA titers. tration of gene therapy-which involves the generation of FIG. 3 is a diagram illustrating the Suppressogenic rela immunogenic material in a patient deficient of the corre tionship between the treatment of (B6D2)F1 mice with sponding gene-possible and effective. OA-mPEG conjugates of different molecular compositions 40 and the primary anti-OA IgG, as measured by radio SUMMARY OF THE INVENTION immunoassay employing the paper radio immunosorbent procedure. The percentages in brackets represent the degree Gene therapy procedures as currently practiced-involve of Suppression with respect to the minimal immune response the administration by itself of a foreign genetic material, or in animals receiving phosphate buffered saline (PBS) in lieu of its biologically active products-do have certain disad 45 of the conjugates. Vantages and limitations which are primarily due to their potential immunogenicity in the host deficient of the corre DESCRIPTION OF THE INVENTION sponding gene. The objectives of the present invention aim at Overcoming the above mentioned complications by Sup The objectives of the present discovery are accomplished pressing the production of antibodies to the foreign thera 50 by a method, wherein an immunosuppressively effective peutic genetic material and of its expression products, and of amount of a tolerogen incorporating a foreign genetic mate thus ensuring the efficacy of gene therapy by the prior rial or its active derivative(s) is administered to the mammal administration of immunosuppressive doses of tolerogenic prior to the administration of the foreign genetic material or conjugates consisting of therapeutically active and poten its biologically active derivative(s). The tolerogenic conju tially immunogenic materials coupled to nonimmunogenic 55 gate is preferably administered to individuals who have not polymers, thus overcoming or minimizing the risk of induc received a prior treatment with the foreign genetic material ing anaphylactic reactions or immune complex diseases. or its product, i.e. to unsensitized individuals. Thus, the main objective of the invention aims at SuppreSS The invention will provide improved methods for gene ing Substantially an immune response to the protein resulting therapy of different human diseases which can be amelio as a consequence of Successful gene therapy, which response 60 rated or eliminated by the administration of the appropriate would undermine the therapeutic efficacy of a biologically genetic materials, etc. or their therapeutic derivatives, of active genetic material and which may also cause untoward which the patient is deficient. The tolerogenic conjugates physiological reactions (e.g. anaphylaxis and/or immune may be Synthesized by covalent or noncovalent attachment complex diseases). of nonimmunogenic polymers to natural or Synthetic bio The invention provides a method for conducting gene 65 logically active proteins Such as for example (i) murine or rat therapy comprising administration to a mammal of an monoclonal antibodies to human T-cells which have been immunosuppressing effective amount of a tolerogenic con used to Suppress transplant rejection (Colvin, R. B. et al., 6,093,699 S 6 Fed. Proc. 41 (1982) p. 363, Abstr. 554) or as “miracle necessary Such groups may have to be introduced into Said bullets” for the destruction of tumors (Froese, G. et al.; molecules before the coupling reaction. Such active groups Immunology 45 (1982) p. 303-12, and Immunological are for example -NH, -NCS, -SH, -OH, -CHI and Reviews 62 (1982), Ed. G. Möller, Munksgaard, Copen COOH and they may be introduced according to well hagen), (ii) enzymes, Such as Superoxide dismutase (Kelly, known methods, if not already present in the molecules used K. et al.; Cdn. J. of Physiol. Pharmacol., 609 (1982) p. for the production of tolerogenic conjugates. 1374-81) or L-asparaginase (Uren, J. r. et al., Canc. In order to minimize the liberation in vivo of the immu Research 39 (1979) p. 1927-33), or (iii) natural or synthetic nogenic and/or allergenic constituent(s) of the tolerogenic hormones. conjugates and to maximize their effectiveness at a low dose, In the presently best developed and therefore also cur it is desirable that the covalent link between the water rently best preferred mode of the invention, the tolerogen is Soluble polymer and protein or its active derivative(s) should a covalent conjugate between monomethoxypolyethylene be as Stable as possible under physiological conditions. glycol (mPEG) with molecular weight in the range of The coupling of the polymer onto the antigenic or genetic 4,500-10,000 daltons and a foreign protein such as ovalbu material must, as mentioned above, have been carried out to min (OA), which served as a model protein. According to Such an extent that the conjugate is rendered tolerogenic, as this modality, tolerogens of appropriate composition (i.e. 15 well as Substantially non-allergenic and Substantially non consisting of the genetic material or its expression product immunogenic. In other words the tolerogens must retain a and an optimal number of mPEG chains attached to it certain number of epitopes of the unmodified antigen, as covalently) Substantially Suppress the formation of antibod long as their immunogenicity has been decreased to that they ies of different classes (e.g. IgE and IgG) which are directed do not induce the formation of antibodies which may cause Specifically against the genetic material perse and/or against unacceptable adverse reactions. its expression product(s). The latter case is exemplified by To achieve tolerogenicity, the degree of Substitution, also OA or its covalent derivative with a number of 2,4- referred to as the degree of conjugation, which is defined as dinitrophenyl groups (DNP), i.e. OA-DNP, where n repre the number of polymer molecules coupled per antigen Sents the average number of DNP groups coupled per one molecule, varies from one antigen molecule to another OA molecule. 25 depending on the nature and size of the antigen and on the Animal Model polymer and its molecular weight. Therefore, for the Syn The acceptability of the mouse as an experimental model thesis of a tolerogenic conjugate of a given antigen it is for correlation to human utility in the present experiments is essential to Synthesize a Series of conjugates with different evidenced by Dreborg et al. “Immunotherapy with degrees of Substitution and then establish the Special range Monomethoxypolyethylene Glycol Modified Allergens”. wherein the above mentioned requirements are fulfilled. Too page 325, which indicates that Similar results were achieved low a degree of Substitution may result in conjugates Still in humans and mice and thus confirms mice are an accept endowed with allergenic and immunogenic properties, and able experimental model for evaluation of mPEG-modified too high a degree of Substitution may result in conjugates allergens. See also Antibodies: A Laboratory Manual, Cold which are not tolerogenic. One of skill in the relevant art will Spring Harbor Press, 1988, p. 93, which indicates that 35 be able to optimize the degree of Substitution using the laboratory mice are an acceptable experimental animal disclosure as example. The optional Substitution range is one model for examining the immune response, and that mice, in in which tolerogenicity is achieved. One of skill in the art particular, possess appropriate characteristics for Studies of can perform the Steps outlined in the Specification and arrive the genetics of the immune response. at the appropriate degree of coupling of the nonimmuno The Tolerogen Employed 40 genic polymer onto the antigenic protein So as to achieve the AS water-Soluble polymers to be used for the preparation claimed properties. of a tolerogen, polyethylene glycols, having molecular In view of the finely tuned homeostatic balance of the weights in the range of 2,000 to 35,000, have proved to be immune response, which may be easily perturbed either effective. Polyethylene glycols in this context also include upwards or downwards by the administration of a given physiologically acceptable derivatives thereof, Such as 45 antigen depending on its dose, State of aggregation and route mono-alkyl ethers, preferably the monomethyl ether, of administration, as well as the presence or absence of whereby the remaining Single terminal hydroxyl groups of adjuvants, it is critical when practicing the invention for the molecules are conveniently used for coupling to the treatment of appropriate disease conditions, that the tolero protein. genic conjugates be administered in Such a manner as to lead Also other water-Soluble polymers (macromolecules) 50 to the down-regulation of the immune response with respect may be used, Such as poly Vinyl alcohols, to one or more classes of immunoglobulins directed against polyvinylpyrrolidones, polyacrylamides and homo- as well the unconjugated biologically active product of the genetic as hetero-polymers of amino acids, polysaccharides (e.g. material. Hence, in practicing this invention for treatment of pullulan, inulin, dextran and carboxymethyl cellulose) or appropriate diseases, the tolerogenic conjugates are to be physiologically acceptable derivatives of these polymers. 55 injected in absence of adjuvants Since the adjuvants may For the covalent coupling of Such polymers to the genetic counteract their SuppreSSogenic effects. However, the inclu material or its antigenic expression molecules, chemical Sion of adjuvants along with the unconjugated immunogenic methods normally used for coupling of biologically active material in the examples given below was justified So as to materials to polymerS may be used. Such methods include Stimulate in experimental animals the enhanced production coupling by means of mixed anhydride, cyanuric chloride, 60 of antibodies in a relatively short time and to thus test under isothiocyanate, reaction between SH derivatives and CHI more Stringent conditions the capacity of the tolerogenic derivatives of the reacting molecules. However, it is obvious conjugates to Suppress the immune response in these ani to the workerS Skilled in the art that other appropriate mals even under these extreme conditions which are par chemical methods may be used to lead to the production of ticularly favorable for enhancing the immune response. conjugates of desired compositions. 65 The Foreign Genetic Material or Vehicle The coupling reaction is made between active groups in In the claims and in the Specifications, proteins and the antigen molecules and in the polymer molecules. If polypeptides are used synonymously. In the present context 6,093,699 7 8 and claims the term foreign genetic material refers to a the protein or to its derivative(s) which will be produced as nucleotide, DNA, RNA, mRNA, plasmid, which are used as a result of the gene therapy. According to one mode of the carriers of the gene and/or the gene itself responsible for the invention, further doses of the tolerogen may be given in expression of the appropriate protein or protein derivative conjunction with the protein or its derivative(s), i.e. after the (fragments included), which are Substantially immunogenic primary administration of the tolerogen. This mode may in the animal to be treated. represent one way of Sustaining the Suppression and may According to one aspect of the invention the genetic offer a more efficient therapeutic regimen for the disease material should be therapeutically effective. Many such condition for which the treatment has been designed. proteins, vectors, DNA are known per se (Culver, K. W., The invention will now be illustrated by some non “Gene Therapy”, 1994, p. xii, Mary Ann Liebert, Inc., limiting examples wherein OA and its tolerogenic mPEG Publishers, New York, N.Y., incorporated herein by refer derivatives have been applied as model Substances to con ence in its entirety). For the purposes of example only, firm the usefulness of the proposed immunosuppressive vectors may be Selected from the group consisting of Molo treatment of a well-established animal model commonly ney murine leukemia virus vectors, adenovirus vectors with utilized in the field of immunology, The conjugates will be tissue specific promoters, herpes simplex vectors, Vaccinia 15 designated as OA-(mPEG), where n represents the average vectors, artificial chromosomes, receptor mediated gene degree of conjugation. FIGS. 1, 2 and 3 show diagrams delivery, and mixtures of the above vectors. Gene therapy illustrating the efficiency of the invention. The percentages vectors are commercially available from different laborato in brackets of FIGS. 1 and 3 represent the degree of ries Such as Chiron, Inc., Emeryville, Calif., Genetic Suppression with respect to the minimal immune response in Therapy, Inc., Gaithersburg, Md., Genzyme, Cambridge, animals receiving phosphate buffered saline (PBS) in lieu of Mass.; SomatX, Almeda, Calif.; Targeted Genetics, Seattle, the conjugates. Wash.; Viagene and Vical, San Diego, Calif. EXAMPLE 1. The effective doses (amounts) and formulations com Preparation of OA-mPEG Conjugates having Different monly used are also known and may be applied to the Degrees of Substitution present invention, although the invention potentially may 25 The conjugates used in the experiments given below have employ reduced or increased doses. In principle, both the been prepared by coupling mPEG molecules to OA essen biologically active foreign genetic material or its tially according to the procedure described by Abuchowski derivatives, as well as the corresponding tolerogenic et al. (J. Biol. Chem. 252, 3518, 1977 utilizing cyanuric conjugates, may be administered parenterally in a Soluble chloride as one of the possible coupling agents. To begin form in isotonic Solution and after removal of aggregates by with, in the experiment described the “active intermediate' centrifugation. Moreover, to destroy unwanted cells, Such as consisting of an mPEG molecule attached to cyanuric chlo cancer cells or the host's cytotoxic cells responsible for ride was prepared. auto-immune diseases, one may insert genetic material con It was found that the most important condition of this sisting in tandem of the DNA specific for the carrier of the reaction was that all reagents be completely anhydrous and bullet (e.g., an antibody molecule directed to a cell marker) 35 that the reaction mixture be protected from atmospheric and the DNA representing the bullet (e.g., toxins represented moisture because of its high Susceptibility to hydrolysis. by ribosome inactivating proteins) into the patient. Among various methods used for the Synthesis of the “active Time Intervals for the Administration intermediate', the example given below illustrates the gen For the induction of immunological tolerance to a given eral procedure. (See also Jackson, C.-J. C., Charlton, J. L., protein the protocol followed according to the invention 40 Kuzminski, K., Lang, G. M. and Sehon, A. H. “Synthesis, comprises the administration initially of an immunosuppres isolation and characterization of conjugates of ovalbumin Sively effective dose (amount) of tolerogen, which is given with monomethoxypolyethylene glycol using cyanuric chlo prior to the administration of the therapeutically active ride as the coupling agent. Anal. Biochem. 165: 114, 1987, protein or its product. If necessary, this dose may be por incorporated herein by reference in its entirety.) tioned and given on repeated occasions. The immunoSup 45 Monomethoxypolyethylene glycol (2.5 g. mol wt 5590, pressive dose which is given may vary from tolerogen to Union Carbide) was dissolved with warming in anhydrous tolerogen, but it has to be administered prior to the entry of benzene (40 ml) and a portion of the benzene (20 ml) was the protein into the host's System. According to the prin removed by distillation to azeotrope off any water in the ciples outlined in the examples, the practitioner Skilled in the polymer. Cyanuric chloride (CNCI), 0.83 g, Aldrich, art can determine the variables Such as dose of tolerogen and 50 recrystallized from benzene was added under nitrogen the minimum interval of time between its administration and followed by potassium carbonate (0.5 g. anhydrous the appearance of the immunogenic protein in the host's powdered) and the mixture stirred at room temperature for System. See, for example, references discussed in back 15 hours. The mixture was then filtered under dry nitrogen ground of the invention. However, it is to be expected that and the filtrate mixed with anhydrous petroleum ether (ca 50 gene therapy, resulting in the production of a “new” protein 55 ml, b.pt. 30–60° C.) in order to precipitate the polymer. The in the protein-deficient patient, has to be preceded by polymer was Separated by filtration under nitrogen, dis administration of the Specific tolerogenic conjugate, i.e., the solved in benzene (20 ml) and reprecipitated with petroleum conjugate comprising the Same protein and capable of ether. This proceSS was repeated Seven times to insure that Suppressing Selectively the immune response of the host the polymer was free of any residual cyanuric chloride. The with respect to the protein in question. 60 active intermediate was finally dissolved in benzene, the A time period of at least one day prior to the administra Solution frozen and the benzene Sublimed away under high tion of the foreign genetic material is preferred, if not even Vacuum to leave a fine white powder. mandatory. In a more preferred embodiment, the tolerogenic Elemental analysis of the intermediate confirmed that it conjugate is administered at least Seven days prior to admin contained 2 chlorine atoms. The intermediate, correspond istration of the foreign genetic material. The immunoSup 65 ing to C2s63Ho77O1272NaCl2. With an average molecular pressive dose refers to the amount of tolerogen required to weight of 5,738 daltons would have a theoretical composi Substantially reduce the immune response of the patient to tion in percentages of C, 53.65; H, 8.92; N, 0.73; Cl, 1.24; 6,093,699 9 10 which agrees with its determined composition of C, 53.51; Thus, whereas treatment of groups of four (B6D2)F1 mice H, 8.89; N, 0.77; C1, 1.08. each with 50 tug of OA-mPEG, or OA-mPEG, or The chloride content of the intermediate was also deter OA-mPEG7 one day prior to intraperitoneal immunization mined by hydrolysis and titration of the chloride released with the Sensitizing dose, consisting of 1 lug of OA and 1 mg with silver nitrate. Thus, the activated intermediate (120 mg) Al(OH), led to essentially complete (99-100%) abrogation was dissolved in water (10 ml) and the pH adjusted to 10 of the primary anti-OA IgE response, as measured-on day with dilute sodium hydroxide. After heating at 90° C. for two 14 after immunization-by PCA in hooded rats, the more hours, the Solution was cooled and the chloride titrated with Substituted conjugates, i.e. O A-mPEGI or and silver nitrate (0.001N), using a chloride ion selective elec OA-mPEG, inhibited the anti-OA IgE response, trode to indicate the endpoint. The chloride content of the 1O respectively, only to the extent of 94% and 50%. In this and activated intermediate was found to be 2.1, consistent with the following examples, the weights of the conjugates given the structure shown above. correspond to their protein content. The OA 40 mg, purified by chromatography on Ultro gel(R) AcA-54 (LKB, Bromma, Sweden) was dissolved in EXAMPLE 3 sodium tetraborate buffer (4 ml, 0.1 M, pH 9.2) and the 15 Long Lasting Suppression of the IgE Response by Protein activated mPEG added to the solution at 4 C. The amount mPEG Conjugates in Contrast to a Transient Suppressive of activated mPEG was varied to prepare conjugates of Effect of Unconjugated Protein differing degrees of polymer Substitution. Mole ratioS It is to be noted that even unmodified OA was capable of (mPEG/OA) used to prepare specific conjugates are given in downregulating the primary IgE response in relation to the Table 1. The polymer-protein mixture was stirred for one response of control mice which had received PBS instead of half hour at 4 C. and then one half hour at room tempera OA or conjugates. In this experiment three groups of four ture. The reaction mixture was desalted by either dialyzing (B6D2)F1 mice each received phosphate buffered saline, or for four days against running distilled water or by passing 50 tug of OA-mPEGs or 50 lug of OA. All animals were bled through a column of Sephadex(R) G-25 (Pharmacia Fine on day 10, 14, 21, 27, 35, 42 and 49 and their IgE titers were Chemicals AB, Uppsala, Sweden). 25 determined by PCA in hooded rats. As illustrated in Table 2, A DEAE-cellulose or DEAE-Sephacryl(R) (Pharmacia it is important to point out that whereas the SuppreSSogenic Fine Chemicals AB, Uppsala, Sweden) column (5 cm by 30 effect of OA-mPEG conjugates was long-lasting, the down cm) was equilibrated with phosphate buffer (0.008 M, pH regulating effect of free OA was of Short duration and, in 7.7). The Salt free OA conjugates were applied in water and actual fact, its administration predisposed the animals to an the free (unbound) mPEG washed through the column with anamnestic response which reached, after booster immuni the pH 7.7 buffer. Free mPEG was detected on thin layer zation (administered on day 28), IgE antibody levels equiva chromatography Camag (Kieselgel DSF-5, Terochem Lab lent to those of control animals which had received PBS and Ltd, Alberta) eluant 3:1 chloroform/methanol using iodine the two sensitizing doses of one antigen. The results given vapor for development. After removal of the free mPEG in Table 2 clearly demonstrate that a tolerogenic conjugate from the ion-exchange column, sodium acetate buffer (0.05 35 injected prior to repeated administration of the correspond M, pH 4.0) was used to elute the conjugate. The conjugate ing free protein essentially abrogated the immune response. fractions were dialyzed and lyophilized to give the dry conjugates. TABLE 2 Effect of administering 50 tug of OA-mPEGs or of free OA TABLE 1. 40 One day prior to immunization Preparation of OA-mPEG, Coniugates PCA titers for groups of Day of bleeding after mice treated with Conjugates' Preparation ratio % mPEGee % OAde primary immunization PBS OA OA-mPEGs OA-mPEGs. 10:1 26 70 45 OA-mPEG 25:1 36 47 1O 5,120 40 <4 OA-mPEG, 25:1 42 47 14 1,940 40 <4 OA-mPEG 50:1 51 41 21 1,280 40 <4 OA-mPEG 50:1 52.4 38 27 640 40 <4 35 1920 1920 160 The degree of substitution, n, is calculated by the formula 50 42 2,560 1,280 160 % mPEG mol wt OA 49 5,120 N.D.* 160 X % OA mol wt mPEG Mole ratio mPeg:OA based on a molecular weight of 5.740 for mPEG On day 28 all three groups received a booster dose of the sensitizing OA dichlorocyanurate and 44.460 daltons for OA. preparation. The percentages of mPEG by weight were determined by nuclear mag N.D. = not determined neticThe percentagesresonance (NMR). of protein by weight were determined by the biuret 55 method. EXAMPLE 4 The total compositions of the conjugates, as calculated form the NMR The Effect of Different Doses of the Tolerogen on the IgE and biuret analysis, are only of the order of 90% of the samples by Response weight; the difference of the order of 10% attributed to moisture absorbed by the conjugates and/or to small amounts of DEAE-cellulose leaching Each OA-mPEG conjugate was injected into groups of 4 from the column. 60 mice each at the four doses of 10 ug, 50 lug, 150 lug and 600 Aug. The control group of mice received PBS as placebo. EXAMPLE 2 AS is evident from FIG. 2, treatment with different con Determination of the Immunosuppressive Effect on the Iq. jugates at doses of 10 ug and 50 ug per mouse revealed Response of Different OA-mPEG, Conjugates marked differences in their Suppressogenic capacity. It is The results of experiments illustrated in FIG. 1 clearly 65 also to be noted that at a dose of 150 lug, all conjugates were demonstrate the Stringent dependency of the SuppreSSoge highly Suppressive and at 600 lig (data note shown) all the nicity of mPEG conjugates on their molecular composition. compounds tested Suppressed completely the IgE response. 6,093,699 11 12 EXAMPLE 5 that pan-specific Suppression of the “human' antibody The Effect of Different Doses of the Tolerogen on the IgE responses against murine monoclonal antibodies (i.e., Response HAMA responses) of the IgG class could be achieved with The Sera used in FIG. 3 to illustrate the effect of tolero 8 tolerogenic mPEG preparations, each consisting of one of genic conjugates on the IgG response were the same as those the 4 gamma chains and of one of the two types of light used in FIG.1. As illustrated in FIG. 3, administration of 50 chains of murine IgG (Bitoh, S., Lang, G. M., Kierek Aug of OA-mPEG7 resulted in the maximal Suppression, i.e. Jaszczuk, D., Fujimoto, S. and Sehon, A. H. Specific immu of the order of 98% of the primary anti-OA IgG response, noSuppression of human anti-murine antibody (HAMA) which was determined 14 days after the first injection of the responses in hu-PBL-SCID mice. Hum. Antibod. Hybrido Sensitizing dose of OA by a radio-immunoassay employing mas 4: 144-151, 1993). the paper radio immunosorbent procedure (Kelly, K. A. et The utility of this technology for therapeutic Strategies in al.; J. Immunol. Meth. 39 (1980) p. 317–33) utilizing OA man, which necessitate the administration of immunogenic bound to the paper and with as I-labelled affinity purified Biological Response Modifiers (BRMs), is apparent. sheep antiserum to mouse IgG. The safety of administration of MPEG conjugates of 15 EXAMPLE 6 different allergenic proteins has been established in clinical The Suppressive Effect of OA-mPEGo on IgM, IgG, and trials in a number of countries in close to 300 patients IgE Plaque forming Cells (PFC) in Spleen and Lymph afflicted by a variety of allergies medicated by IgE antibod Nodes. ies (Dreborg, S. and Akerblom, E. Immunotherapy with One mg of OA-mPEGo (containing 10 mPEG groups monomethoxypolyethylene glycol modified allergens. In: S. with an average mol wt of 10,000 daltons, which were D. Bruck (Ed.), CRC Crit. Rev. Ther. Drug Carrier Syst. coupled per OA molecule by the Succinic anhydride method 6:315–363, (1990)). (Wie, S. I. et al., Int. Archs. Allergy appl. Immun. 64, 84 It is to be emphasized that the function of the mPEGylated (1981)) or PBS was administered intraperitoneally to each protein in the present Strategy is to induce Suppressor T (Ts) group of four (B6D2)F1 mice each one day prior to immu 25 cells which recognize the epitopes shared by both the nization with 1 lug of DNP-OA in 1 mg Al(OH). unmodified and the mPEGylated BRM. In other words, this On Several days thereafter the Spleen, as well as the technology leads to conversion of antigens not only to mesenteric, parathymic and inguinal lymph nodes were nonimmunogenic, but most importantly to actively immu removed and assayed for IgM, IgG, and IgE anti-DNP PFC noSuppressive molecules, which induce immunologic toler (Rector, E. S. et al., Eur. J. Immunol. 10, p. 944-49 (1980). ance with respect to the original unmodified protein antigen. In Table 3 are given the numbers of PFC in the above tissues By contrast, the purpose of Some other workers and com 10 days after immunization; from these data it is evident that panies utilizing mPEG conjugates of BRMs is to only reduce treatment with this tolerogen markedly reduced the number their immunogenicity i.e., without converting them to active of IgM, IgE, and IgG PFC in all tissues examined. tolerogens, and to thus only increase their half-life in cir Therefore, these results Support the claim that the tolerogens 35 culation. shut off the immune response rather than neutralize the The discovery that pretreatment of a host with tolerogenic circulating antibodies. mPEG conjugates of a given protein Ag, followed by administration of the unmodified Ag, results in abrogation of TABLE 3 the host's capacity to mount an antibody response to the Ag 40 in question has a direct utility in Some forms of "gene The effect of OA-mPEGo on the suppression of IgM, IgG and IgE therapy” which would result in the production of the protein plaque forming cells (PFC) in Spleen and lymph nodes corresponding to the gene in question, on condition that the Anti-DNP PFC per 10 cells from different tissues" host would have been deficient of the gene responsible for the expression of the particular protein from birth (see, Antibody Parathymic Mesenteric Inguinal 45 Sehon, A. H. Suppression of antibody responses by chemi Class Treatment Spleen Nodes Nodes Nodes cally modified antigens, Carl Prausnitz Memorial Lecture, IgM PBS 2,150 2,950 Nd Nd:* * XVIII Symposium Collegium Internationale OA-mPEG 900 2OO Nd Nd IgG PBS 15,350 78,550 5,000 Nd Allergologicum, Int. Arch. Allergy appl. Immunol.94:11-20, OA-mPEG Nd 1,300 Nd Nd 1991; Takata, M., Maiti, P. K., Kubo, R. T., Chen, Y-H. IgE PBS 10,410 16,530 11,140 3OO 50 Holford-Stevens, V., Rector, E. S. and Sehon, A. H. Cloned OA-mPEG 500 950 400 Nd Suppressor T cells derived from mice tolerized with conju gates of antigen and monomethoxypolyethylene glycol. J. *Each tissue sampling represents a pool from 4 mice **Nd = undetected Immunol. 145:2846-2853, 1990; and Takata, M., Maiti, P. K., Bitoh, S., Holford-Stevens, V., Kierek-Jaszczuk, D., The above experiments establish the immunosuppressive 55 Chen, Y., Lang, G. M. and Sehon, A. H. Downregulation of effects discussed above and the effects at the various doS helper T cells by an antigen-Specific monoclonal TS factor. ageS. Cell. Immunol. 137:139-149, 1991). On the basis of well known immunological principles, it EXAMPLE 7 would be obvious that in conditions when the host is totally In addition, utilizing the hu-PBL-SCID mice, it was 60 deficient, from birth, of the gene which has to be inserted demonstrated that in accordance with the phenomenon of after the maturation of the immune System, the protein “linked immunological Suppression', croSS-Specific Sup expressed by the gene in question induces in the host an pression of the human antibody response could be induced immune response against itself, (since the host with a normal to murine mAbs which differ in their antigen binding Speci immune system would not have been rendered from birth ficities from those of the murine is which had been incor 65 tolerant to the protein in question). Thus, (i) the immune porated into the tolerogenic conjugates, on condition that response of the host to this protein would be manifested in both mAbs shared the same heavy and light chains. Thus, the production of antibodies or cytotoxic cells by the host to 6,093,699 13 14 the “new” protein, and (ii) this immune response would not mPEG is detected on thin layer chromatography Camag only neutralize the “new” protein, but may also lead to (Kieselgel DSF-5, Terochem Lab Ltd, Alberta) eluant 3:1 diverse therapeutic complications due to formation of chloroform/methanol using iodine vapor for development. “immune complexes’ consisting of the resulting antibody After removal of the free mPEG from the ion-exchange antigen aggregates. column, sodium acetate buffer (0.05 M, pH 4.0) is used to Clearly, the one condition for which gene therapy has elute the conjugate. The conjugate fractions are dialyzed and proven to be an effective therapeutic modality is adenosine lyophilized to give the dry conjugates. deaminase (ADA) deficiency, which results in the impair ment of T lymphocytes and hence in the Severe combined Conjugates of the CFTR gene are administered to a immunodeficiency disorder (SCID) in children deficient of patient at least one day prior to transfer of the cystic fibrosis ADA from birth. Therefore, it is not surprising that Dr. transmembrane conductance regulator gene to lung tissue Culver was Successful in developing a curative gene therapy using recombinant adenoviral vectors or liposomes. for this condition by treating the SCID kids by infusion of their own “ADA gene-corrected cells”. However, unless gene therapy is instituted from birth, 15 EXAMPLE 10 when it is relatively easier to induce immunological toler ance to a foreign genetic material or its expressed products, The expressed protein material of the low density lipo than in adulthood after maturation of the immune System, protein receptor (LDLr) gene used in the treatment of the Success of gene therapy in hosts with a well formed familial hypercholesterolemia is dissolved in Sodium tet immune system would be undermined by the above raborate buffer (4 ml, 0.1 M, pH 9.2) and the activated mentioned complications. Hence, to avoid these mPEG added to the solution at 4 C. The amount of activated complications, the induction of immunological tolerance to mPEG is varied to prepare conjugates of differing degrees of a well-defined protein Ag, by pretreatment of the host with polymer substitution. Different mole ratios (mPEG/gene tolerogenic mPEG conjugates of the corresponding Ag, i.e., product) is used to prepare specific tolerogenic conjugates as Ag(mPEG), is indispensable for the Success of gene 25 described earlier. The polymer-gene product mixture is therapy in disease conditions, when the same protein is stirred for one half hour at 4 C. and then one half hour at expressed by the inserted gene, Since this protein would room temperature. The reaction mixture is desalted by either induce deleterious immune response against itself in the dialyzing for four days against running distilled water or by host. passing through a column of Sephadex(R) G-25 (Pharmacia The present invention is the confirmation and extension of Fine Chemicals AB, Uppsala, Sweden). the earlier discovery of induction of immunological toler ance by immunosuppressive Ag(mPEG), conjugates to A DEAE-cellulose or DEAE-Sephacryl(R) (Pharmacia therapies involving the insertion of new genes into the host Fine Chemicals AB, Uppsala, Sweden) column (5 cm by 30 with an intact immune System. cm) is equilibrated with phosphate buffer (0.008 M, pH 7.7). 35 The salt free mPEG conjugates of the LDLr-gene products EXAMPLE 8 are applied in water and the free (unbound) mPEG washed Thus, in accordance with the present invention, prior to through the column with the pH 7.7 buffer. Free mPEG is beginning of gene therapy, i.e., prior to insertion of a new detected on thin layer chromatography Camag (Kieselgel gene into a host which is required for expression of a protein 40 DSF-5, Terochem Lab Ltd, Alberta) eluant 3:1 chloroform/ beneficial to the host, e.g., one of the deficient clotting methanol using iodine vapor for development. After factors or enzymes, it is essential to render the host tolerant removal of the free mPEG from the ion-exchange column, to the protein in question by the use of the invention sodium acetate buffer (0.05 M, pH 4.0) is used to elute the described. conjugate. The conjugate fractions are dialyzed and lyo EXAMPLE 9 45 philized to give the dry conjugates. The expressed protein material of the cystic fibrosis Conjugates of the LDLr gene product are administered to transmembrane conductance regulatory gene (CFTR) a patient. Hepatocytes are grown in the laboratory and (Genzyme, Cambridge, Mass.) for the treatment of cystic genetically altered with a murine retroviral vector containing fibrosis is dissolved in sodium tetraborate buffer (4 ml, 0.1 50 LDLr gene. The cells are reinfused through the hepatic M, pH 9.2) and the activated mPEG added to the solution at artery to the liver of the patient at least one day after 4 C. The amount of activated mPEG is varied to prepare administration of the conjugate. conjugates of differing degrees of polymer Substitution. Table 4 shows a list of gene therapy Systems which have Different mole ratios (mPEG/gene product) are used to been approved by the Recombinant DNA Activities Com prepare specific tolerogenic conjugates as described earlier. 55 The polymer-gene product mixture is stirred for one half mittee of the National Institutes of Health. However, no hour at 4 C. and then one half hour at room temperature. consideration appears to have been given to overcoming the The reaction mixture is desalted by either dialyzing for four potential complications due to the host mounting an immune days against running distilled water or by passing through a response against the respective gene products. Clearly, if the column of SephadeX(R) G-25 (Pharmacia Fine Chemicals 60 patient had been producing from birth these proteins, he/she AB, Uppsala, Sweden). would be tolerant to them, i.e., and gene therapy would not A DEAE-cellulose or DEAE-Sephacryl(R) (Pharmacia necessitate the Strategy of the described invention if the Fine Chemicals AB, Uppsala, Sweden) column (5 cm by 30 patient has retained his/her tolerance between the Shutting cm) is equilibrated with phosphate buffer (0.008 M, pH 7.7). off of his/her own genes producing the desired protein and The salt free mPEG conjugates of the cystic fibrosis gene 65 the time of initiation of gene therapy by transfer of the gene product are applied in water and the free (unbound) mPEG in association with an appropriate "vehicle' for the renewed washed through the column with the pH 7.7 buffer. Free production of the protein. 6,093,699 15 16

Disorder (gene used) Cells altered (vector) Disorder Location Adenosine deaminase deficiency T-cells and stem cells (retroviral) A. Brain tumors (MDR-1) Stem cells (retroviral) 11-Beta-hydroxysteroid dehydrogenase deficiency (1) Chr.1 Brain tumors (primary and meta Tumor cells (retroviral) 3-Beta-hydroxysteroid dehydrogenase, type II, p13.1 static) (HS-tk) deficiency (3) types (1) Brain tumors (primary) (HS-tk) Tumor cells (retroviral) 3-Hydroxyacyl-CoA dehydrogenase deficiency (1) Chr. 7 Brain tumors (primary and meta Tumor cells (retroviral) 3-Ketothiolase deficiency (1) 1q22.3-q23.1 static) (HS-tk) Aarskog-Scott syndrome (2) Xp11.21 Brain tumors (primary and meta Tumor cells (retroviral) 2Abetalipoproteinemia (1) static) (HS-tk) Acanthocytosis. one form (1) Brain tumors (primary) (anti-sense Tumor cells (DNA transfection) Acatalasemia (1) IGF-1) Acetyl-CoA carboxylase deficiency (1) Brain tumors (primary) (HS-tk) Tumor cells (retroviral) 15 Acid-maltase deficiency, adult (1) Brain tumors (primary and meta Tumor cells (retroviral) Acoustic neuroma (2) static) (HS-tk) Acrocallosal syndrome Breast cancer (IL-4) Fibroblasts (retroviral) ACTH deficiency (1) Breast cancer (MDR-1) Stem cells (retroviral) Acyl-CoA dehydrogenase, long chain, deficiency of (1) Breast cancer (MDR-1) Stem cells (retroviral) Acyl-CoA dehydrogenase, medium chain, deficiency Colorectal cancer (IL-4) Fibroblasts (retroviral) of (1) Colorectal cancer (IL-2 or TNF-C. Tumor cells (retroviral) Acyl-CoA dehydrogenase, short chain, deficiency of (1) gene) Adenylosuccinase deficiency (1) Colorectal cancer (HLA-B7 and Tumor cells (liposomes) Adrenal hyperplasia, congenital, due to 11-beta-hydroxy f2-microglobulin) lase deficiency (1) Colorectal cancer (IL-2) Fibroblasts (retroviral) Adrenal hyperplasia, congenital, due to 21-hydroxylase Cystic fibrosis (CFTR) Respiratory epithelium (adenoviral) deficiency (3) Cystic fibrosis (CFTR) Respiratory epithelium (adenoviral) 25 Adrenal hyperplasia V (1) Cystic fibrosis (CFTR) Respiratory epithelium (liposomes) Adrenal hypoplasia, primary (2) Cystic fibrosis (CFTR) Respiratory epithelium (adenoviral) Adrenocortical carcinoma (2) Cystic fibrosis (CFTR) Respiratory epithelium (adenoviral) Adrenocortical carcinoma, heriditary (2) Cystic fibrosis (CFTR) Respiratory epithelium (adenoviral) Adrenoleukodystropy (2) Familial hypercholesterolemia Liver cells (retroviral) Adrenomyeloneuropathy (2) (LDLr) APP deficiency, congenital (1) Gaucher disease' (glucocere Stem cells (retroviral) Agammaglobulinemia, type 1, X-linked (3) brosidase) Agammaglobulinemia, type 2, X-linked (2) Gaucher disease' (glucocere Stem cells (retroviral) Aicardi syndrome (2) brosidase) agile syndrome (2) Gaucher disease' (glucocere Stem cells (retroviral) binism (3) brosidase) 35 btalma, brown, 203294 (1) Gaucher disease (glucocere Stem cells (retroviral) binism, oculoeutaneous, type II (3) brosidase) binism-deafness syndrome (2) HIV infection (Mutant Rev) T cells (retroviral) I bright hereditary Osteodystrophy-2 (2) HIV infection (HIV-1 III env) Muscle (retroviral) AI dolase A deficiency (1) HIV infection (HIV-1 IIIB Env and Muscle (retroviral) dosteroslam, glacocorticoid-remediable (1) Rev) lan-Herndon syndrome (2) HIV infection (HIV-1 ribozyme) T cells (retroviral) 40 pha-1-antichymetrypsin deficiency (1) Leptomeningeal carcinomatosis Tumor cells (retroviral) pha-NAGA deficiency (1) (HS-tk) pha-thalassemia/mental retardation syndrome, Malignant melanoma (IL-4) Tumor cells (retroviral) pe 1 (1) Malignant melanoma (IL-2) Tumor cells (retroviral) pha-thalassemia/mental retardation syndrome, Malignant melanoma (IL-2) Tumor cells (retroviral) pe 2 (2) Malignant melanoma (IL-2) Tumor cells (retroviral) 45 port syndrome, 301050 (3) Malignant melanoma (IL-4) Fibroblasts (retroviral) veolar proteinois, congenital, 265120 (1) Malignant melanoma (HLA-B7) Tumor cells (liposomes) zheimer disease, APP related (3) Malignant melanoma (HLA-B7 Tumor cells (liposomes) Alzheimer disease-1 (2) and f-microglobulin) zheimer disease-3 (3) Malignant melanoma (TNF-ct, or T cells or tumor cells (retroviral) A zheimer disease-2, late onset (2) IL-2) 50 Amelogenesis imperfecta (1) Malignant melanoma (interferon-Y) Tumor cells (retroviral) Amelogenesis imperfecta-3, hypoplastic type (2) Malignant melanoma (B7) Tumor cells (retroviral) AMP deaminase deficiency, erythrocyte (1) Neuroblastoma (IL-2) Tumor cells (retroviral) Amyloid neuropathy, familial, several allelic Non-small cell lung cancer (p53 or Tumor cells (retroviral) Amyloidosis, cerebroarterial, Dutch type (1) antisense K-ras) Amyloidosis, Finnish type, 105120 (1) Ovarian cancer (HS-tk) Tumor cells (retroviral) 55 Amyloidosis, hereditary renal, 105200 (1) Ovarian cancer (MDR-1) Stem cells (retroviral) Amyloidosis, Iowa type, 107680.0010 (1) Ovarian cancer (MDR-1) Stem cells (retroviral) Amyloidosis, secondary, susceptibility to (1) Renal cell carcinoma (IL-2) Tumor cells (retroviral) Amyloidosis, senile systemic (1) Renal cell carcinoma (IL-4) Fibroblasts (retroviral) Amyotrophic lateral scierosis, juvenile (2) Renal cell carcinoma (TNF-ct, or Fibroblasts (retroviral) Amytrophic lateral scierosis, one form, 105400 (3) IL-2) Anal canal carcinoma (2) Renal cell carcinoma (GM-CSF) Tumor cells (retroviral) 60 Analbuminemia (1) Small cell lung cancer (IL-2) Tumor cells (DNA transfection) Anemia, megalobiastic, due to DHFR Solid tumors (HLA-B7 and B2 Tumor cells (liposomes) deficiency (1) microglobulin) Anemia, pernicious, congenital, due to Chr.11 deficiency of intrinsic factor (1) Anemia, sideroblastic, with spinocerebellar Table 5 shows a list of health disorders for which chro 65 ataxia (2) mosomal locations are known which are considered treat Anemia, sideroblastic/hypochromic (3) Xp11.21 able by gene therapy. 6,093,699 17 18

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Disorder Location Disorder Location Aneurysm, familail, 100070 (1) Central core disease of muscle (2) 19q13.1 Angelman syndrome (2) Centrocytic lymphoma (2) 11q13 Angiodema, hereditary (1) Cerebral amyloid angiopathy (1) 20p11 Anhidrotic ectodermal dysplasia (2) Cerebral arteriopathy with subcortical infarets and 19q12 Aniridia of WAGR syndrome (2) leukoencephalopathy (2) Aniridia-2 (3) Cerebrotendinous xanthomatosis (2) 2q33-qter Ankylosing spondyitis (2) Ceroid lipofuscinosis, neuronal-1, infantile (2) 1p32 Anophthalmos-1 (2) Cervical carcinoma (2) 11q13 Anterior segment mesenchymal dysgenesis (2) CETP deficiency (1) 16q21 Antithrombia III deficiency (3) Charcot-Marie-Tooth neuropathy, slow nerve conduction 17p11.2 ApoA-I and apaC-III deficiency, combined (1) type Ia (2) Apollipoprotein B-100, defective (1) Charcot-Marie-Tooth neuropathy, slow nerve conduction Apollipoprotein H deficiency (1) 15 type Ib (2) Argininemia (1) Charcot-Marie-Tooth neuropathy, X-linked-1, dominant Argininosuccinicaciduria (1) (2) Aspartylglucosaminuria (3) Charcot-Marie-Tooth neuropathy, X-linked-2, Xp22.2 Ataxia-telangiectasia (2) recessive (2) Atherosclerosis, susceptibility to (2) Cholesteryl ester storage disease (1) Atherosclerosis, susceptibility to (3) Chondrodysplasia punctata, rhisomellic (2) Atopy (2) Chrondrodysplasia punctata, X-linked dominant (2) Atransferrinemia (1) Chrondrodysplasia punctata, X-linked recessive (2) 22.3 Atrial septal defect, secundum type (2) Choroideremia (2) Autonomic failure due to DBH deficiency (1) Chronic granulomatose disease, autosomal, due to B deficiency of CYBA (3) Chronic granulomatous disease due to deficiency of 7q11.23 Basal cell nervous syndrome (2) 25 MCF-1 (1) Battern disease (2) Chronic granulomatous disease due to deficiency of ?Battern disease, one form, 204200 (1) NCF-2 (1) Becker muscular dystrophy (3) Chronic granulomatous disease, X-linked (3) b21.1 Beckwith-Wiedemann syndrome (2) Chronic infection, due to opsanin defect (1) Bernard-Soulier syndrome (1) Citrullinemia (1) Biepharaphimosis, spicanthus inversus and Cleft palate, X-linked (2) ptosis (2) Cleidocranial dysplasia (2) Bloom syndrome (2) CMO II deficiency (1) Borjeson-Forssman-Lehmann syndrome (2) Cockeye syndrome-2, late onset, 215410 (2) Bornholm eye disease (2) Coffin-Lowry syndrome (2) p22.2-p22.1 Branchiootic syndrome (2) Colon cancer, familial, nonpolyposis type 1 (2) 2Breast cancer (1) 35 Colorblindness, blue monochromatic (3) Breast cancer (1) Colorblindness, deutan (3) Breast cancer, ductal (2) Colorblindness, protan (3) Breast cancer, ductal (2) Colorblindness, tritan (2) Breast cancer-1, early onset (2) Colorectal adenoma (1) 12p12.1 Brody myopathy (1) Colorectal cancer (1) 12p12.1 Burkitt lymphoma (3) 40 Colorectal cancer (1) 18q23.3 C Colorectal cancer (1) Colorectal cancer, 114500 (3) 2Cld deficiency (1) 1p36.3-p34.1 Colorectal cancer (3) Clr/Cls deficiency, combined (1) 12p13 Combined C5/C7 deficiency (1) C2 deficiency (3) 6p21.3 Combined variable hypogammaglobulinemia (1) C3 deficiency (1) 19p13.3-p13.2 Congenital bilaterial absence of vas deference (1) C3b inactivator deficiency (1) 4q25 45 2Conotruncal cardiac anomellen (2) C4 deficiency (3) Contractural arachnodactyly, congenital (3) C5 deficiency (1) Coproparphyria (1) C6 deficiency (1) Cornelia de Lange syndrome (2) C7 deficiency (1) Coronary artery disease, susceptibility to (1) C8 deficiency, type I (2) 1p32 Cortisol resistance (1) C8 deficiency, type II (2) 1p32 50 CR1 deficiency (1) C9 deficiency (1) 5p13 Craniofrontossal dysplasia (2) pter-p22.2 Campomelic dysplasia-1 (2) 17q24.3-q25.1 Craniosynostosis, type II (2) Carbamoylphosphate synthelase I deficiency (1) 2p Craniosynostosis, type I (2) 7p21.3-21.2 Carbonic anhydrase I deficiency (1) Creatine kinase, brain type, extopic expression Carboxypeptidase B deficiency (I) Chr.13 of (2) Cardiomyopathy (1) 55 Creutzfeldt-Jakob disease, 123400 (3) 20pter-p12 Cardiomyopathy, dilated, X-linked (I) Xp21.2 Crigier-Najjar syndrome, type 1, 218800 (1) hr.2 Cardiomyopathy, familial, hypertrophic, 1, 192600 Cryptorchidism (2) 21 Cardiomyopathy, familial hypertrophic, 2 (2) Cutis laxa, marfanoid neonatal type (1) 7q31.1-31.3 Cardiomyopathy, familial hypertrophic, 3 (2) Cystathioninuris (1) hr.16 Cardlage-hair hypoplasia (2) Cystic fibrosis (3) Cat-eye syndrome (2) Cystimaria (2) Cataract, anterior polar, I (2) 60 Cystimaria, 22O100 (1) 2pter-q32.3 Cataract, congenital total (2) Cataract, congenital, with microphthalmia (2) Cataract, Coppock-like (3) Deafness, conductive with stapes fixation (2) Cataract, Maerner type (2) Deafness, low tone (2) Cataract, Zonular pulverulent-1 (2) Debrisoquine sensitivity (1) CD3, zeta chain, deficiency (1) 1q23-q2521.1 65 Dentinogenesis imperfecta-1 (2) Central core disease (3) 14q12 Denys-Drash syndrome (1) 6,093,699 19 20

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Disorder Location Disorder Location Diabetes insipidus, nephrogenic (3) F Diabetes insipidus, neurohypophyseal, 125700 (1) 2Diabetes mellitus, insulin-dependent-1 (2) Fabry disease (3) Diabetes mellitus, insulin-resistant, with acanthosis Facioscapulohumeral muscular dystrophy (3) nigricans (1) Factor H deficiency (1) Diabetes mellitus, rare form (1) Factor V deficiency (1) Diastrophic dysplasia (2) Factor VII deficiency (1) DiGeorge syndrome (2) Factor X deficiency (1) Diphenylhydantoin toxicity (1) Factor XI deficiency (1) Diphtheria, susceptibility to (1) Factor XII deficiency (1) DNA ligase 1 deficiency (1) Factor XIIIA deficiency (3) 2Dubin-Johnson syndrome (2) Factor XIIIB deficiency (1) Duchenne muscular dystrophy (3) 15 Familial Mediterranean fever (2) Dysalbuminemic hyperthyroxinemia (1) ?Fanconi anemia (1) Dysalbuminemic hyperZincemia (1) Fanconi anemia (2) Dysantonomia, familial (2) Favism (1) Dysfibrinogenemia, alpha types (1) I?Fetal alcohol syndrome (1) Dysfibrinogenemia, beta types (1) Fetal hydantoin syndrome (1) Dysfibrinogenemia, gamma types (1) Fibrodysplasia Ossificans progesssiva (1) Dyskeratosis congenita (2) Fish-eye disease (3) Dyslexia-1 (2) Fish-odor syndrome (1) Dysplaminogenemic thrombophilia (1) Fletcher factor deficiency (1) Dysprothrombinemia (1) Focal dermal hypoplasia (2) Dystransthyretinemic hyperthyroxinemia (1) Friedreich ataxia (2) E Fructose intolerance (1) 25 Fucosidosis (1) 2EEC syndrome (2) Fumarase deficiency (1) Ehlers-Danlos syndrome, type IV, 130050 (3) Ehlers-Danlos syndrome, type VI, 225400 (1) Ehlers-Danlos syndrome, type VIIA1, 130060 (3) G6PD deficiency (3) Galactokinase deficiency (1) Ehlers-Danlos syndrome, type VIIA2, 130060 (3) Galactose epinerase deficiency (1) Ehlers-Danlos syndrome, type X (1) Galactosemia (1) Elliptocytosis, Malaysian-Melanesian type (1) Galactosialidosis (1) Elliptocytosis-1 (3) Gardner syndrome (3) Elliptocytosis-2 (2) Gaucher disease (1) Elliptocytosis-3 (2) Gaucher disease, variant form (1) Emery-Dreifuss muscular dystrophy (2) 35 Genitourinary dyplasia (2) Emphysema (1) Gerstmann-Straussler disease, 137440 (3) Emphysema due to alpha-2-macroglobuline Gilbert syndrome, 143500 (1) deficiency (1) Glanzmann thrombasthenis, type A (1) Emphysema-cirrosis (1) 4.d32.1 Glanzmann thrombasthenia, type B (1) Endocaridal fibrolastosis-2 (2) Xq28 Glaucoma, congenital (2) Enolase deficiency (1) pter-p36.13 Glaucoma, primary open angle (2) Ensinophilic myeloproliferative disorder (2) 40 Glioblastoma multiforme (2) pidermalysis bullosa dystrophica, dominant, 131750 (3) Glucose/galactose malasorption (1) pidermalysis bullosa dystrophica, recessive, 226600 (3) Glutaricacidemia type IIC (3) pidermalysis bullosa, Ogna type (2) Glutaricaciduna, type IIA (1) pidermalysis bullosa simplex, 131900 (3) Glutaricaciduris, type IIB (2) pidermalysis bullosa simplex, Dowing-Meara type, Glutathioninuria (1) 8 1. 7 60 (3) 45 Glycerol kinase defcicency (2) pidermalysis bullosa simplex, Dowing-Meara type, Glycogen storage disease III (3) 1. 7 60 (3) Glycogen storage disease VII (1) pidermalysis bullosa simplex, generalized 131900 (1) Glycogen storage disease, X-linked hepatic (2) Epidermalysis bullosa simplex, localized 131800 (1) Glyoxalase II deficiency (1) Epidermalysis bullosa, Weber Cockayne type, GM1-gangliosidosis (1) i 31800 (2) 50 GM2-gangliosidosis, AB variant (1) pidermolytic hyperkeratosis, 118800 (1) GM2-gangliosidosis, juvenile adult (1) pidermolytic hyperkeratosis, 118800 (3) Goeminne TKCR syndrome (2) pidermolytic palmophantar keratoderma (2) Goiter, adolescent, multinodular (1) pilepsy, genign neonatal (2) Goiter, nonendemic, simple (1) pilepsy, juvenile myoclonic (3) Goldenhar syndrome (2) pilepsy, progressive myoclonic (2) 55 Gonadal dysgenesis, XY female type (2) pithelioma, self-healing squamous 1, Gonadal dysgenesis, XY type (1) Ferguson-Smith type (2) Gonadoblastoma (2) Erythremia (1) Gonadotropin deficiency (2) Erythremias, alpha- (1) 16pter-p13.3 Greig craniopolysyndactyly syndrome (3) Gynecomastia, familial, due to increased Erythremias, beta- (1) 11p15.5 aromatase activity (1) Erythroblastosis fetalis (1) 1p36.2-p34 60 Gyrate atrophy of choroid and retina with Erythrocytosis, familial, 133100 (2) 19p13.3-p13.2 ornithinemia, B6 responsive or unresponsive (1) Erythrokeratodermia varibilis (2) 1p36.2-p34 H Euthyroidal hyper and hypothyrazinemia (1) Xq22 Ewing sarcoma (3) 22q12 Harderoporphyrinuria (1) Chr.9 Exertional myoglobinuris due to deficiency 11p15.4 Harthup disease, 234500 (1) 2pter-q32.3 of LDH-A (1) 65 Heinz body anemias, alpha- (1) 16pter-p13.3 Exadative vitreoretinopathy, X-linked (2) Xq21.31 Heinz body anemias, beta- (1) 11p15.5 6,093,699 21 22

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Disorder Location Disorder Location Hemochromatosis (2) 6p21.3 Hypoalphalipoproteinemia (1) Hemodialysis-related amyloidosis (1) 15q21-q22 Hypobetalipoproteinemia (1) Hemolytic anemia due to ADA excess (1) 20q13.11 Hypocaleturic hypercalcemia, type II (2) Hemolytic anemias due to adenylate kinase 3.q.34.1 Hypoceruloplasminemia, hereditary (1) deficiency (1) Hypofibrinogenemia, gamma types (1) Hemolytic anemias due to bisphosphoglycerate Hypoglycemia due to PCK1 deficiency (1) mutase deficiency (1 Hypogonadism, hypergonadoptropic (1) Hemolytic anemias due to G6PD deficiency (1) Xq23 Hypogonadism, hypogonadotropic due to GNRH Hemolytic anemias due to glucosphosphate 19q13.1 deficiency, 227200 (1) isomerase deficiency (1) Hypomangesemia, X-linked primary (2) Hemolytic anemias due o gluathione peroxidase Hypomelanosis of Ito (2) deficiency (1) Hypomelanosis of Ito (2) Hemolytic anemias due to glutathione reductase 8p21.1 15 Hypoparathyrodism, familial (1) deficiency (1) Hypoparathyrodism, X-linked (2) Hemolytic anemias due to hexokinase deficiency (1) Hypophosphatasia, adult, 1483.00 (1) Hemolytic anemias due to PKG deficiency (1) Hypophosphatasia, infantile, 241500 (3) Hemolytic anemias due to phosphofructokinase Hypophosphatemia, hereditary (2) deficiency (1) Hypophosphatemia with deafness (2) Hemolytic anemias due to triosephosphate isomerase Hypoprothrombinemia (1) deficiency (1) Hypospadias-dysphagia syndrome (2) Hemophilia A (3) Hypothyroidism, hereditary congenital (1) Hemophilia B (3) Hypothyroidism, nongoitrous (1) Hemorrhagic diathesis due to “antithrombin’ Hypothyroidism, nongoitrous, due to TSH resistance (1) Pittsburgh (1) Hemorrhagic diathesis due to PA11 deficiency (1) Hepatic lipase deficiency (1) 25 ?Ichthyosis vulgaris, 146700 (1) q21 Hepatocarcinoma (1) Ichthyosis, X-linked (3) 22.32 Hepatocellular carcinoma (3) ?Immotile cillia syndrome (2) p Hereditary persistance of alpha-fetoprotein (3) 4 Immunodeficiency, X-linked, with hyper-IgM (3) p24-q27 ?Hereditary persistance of fetal hemoglobin (3) Incontinentia pigmenti, familial (2) p27-q28 ?Hereditary persistance of fetal hemoglobin, Incontinentia pigmenti, sporadic type (2) p11.21 heterocellular, indian type (2) Infertile male syndrome (1) scen-q22 ?Hereditary persistence of fetal hemoglobin, Xp11.23 Inosien triphosphatase deficiency (1) 20p Swiss type (2) Insomnia, fatal familial (3) 20pter-p12 ?Hermansky-Padiak, syndrome, 203300 (1) ?Insulin-dependent diabetes meltus-2 (2) 11C Hers disease, or glycogen storage disease VI (1) Interferon, alpha, deficiency (1) 9p21 Heterocellular hereditary persistence of fetal Interferon, immune, deficiency (1) 12q24.1 hemoglobin (2) 35 ?Isolated growth hormone deficiency due to defect in 20p11.23-qter Hex A pseudodeficiency (1) GHRF (1) HHH syndrome (2) Isolated growth hormone deficiency, IIIig type with Histidinemia (1) absent GH and Kowarski type with bioinactive GH (3) Holoprosencephaly, type 3 (2) Isovalericaciidemia (1) Holoprosencephaly-1 (2) Holoprosencephaly-2 (2) Holoprosencephaly-4 (2) 40 ?Jacobsen syndrome (2) Holt-Oram syndrome (2) Holt-Oram syndrome (2) Homocystinuria, B6-responsive and nonresponsive Kallmann syndrome (2) Xp22.3 types (1) Kappa light chain deficiency (1) 2p12 HPFH, deletion type (1) Keratoxis follicularis spinulosa decalvans (2) Xp22.2-p21.2 HPFH, nondeletion type A (1) 45 Kiniogen deficiency (1) 3q26-qter HPFH, nondeletion type G (1) ?Kippel-Feli syndrome (2) 5q11.2 HPRT-related gout (1) Kniest, dysplasia (1) 12q13.11-q13.2 Humoral hypercalcentria of malignancy (1) 2KOstmann agranulocytosis (2) 6p21.3 Huntington disease (2) Krabbe disease (1) 14q24.3-q22.1 Hurler syndrome (1) Hurler-Scheie syndrome (1) 50 Hydrocephalms due to aqueducts of Syivius, 307000 (3) ?Lactase deficiency, adult 223100 (1) Chr.2 Hydrops fetalis, one form (1) Lactase deficiency, congenital (1) Chr.2 Hyperammonemia due to CTPase deficiency (1) ?Lactoferrin-deficient neutrophilis, 245480 (1) 3q21-q23 Hyperbetalipoproteinemia (1) Langer-Giedion syndrome (2) 8q24.11-24.13 Hypercalcemia, hypocalciaric, familial (2) Langer-Saldino achondrogenesis 12q13.11-q13.2 Hypercholesteroliemia, familial (3) 55 hypochrondrogenesis (1) 2Hperglycinemia, isoloated nonketotic, type 1 (2) Laron dwarfism (1) Hyperimmunoglobulin G1 syndrome (2) 2Laryngeal adductor paralysis (2) Hyperkalemic periodic paralysis (3) Lead poisoning, susceptibility to (1) Hyperleucinemia-isoleucinemia or hypervalinemia (1) Leiomyomata, multiple hereditary cutaneous (2) Hyperlipoproteinemia (1) Leiomomatosis-nephropathy syndrome, 308940 (1) Hyperlipoproteinemia, type 1b (1) Leprechanism (1) Hyperlipoproteinemia, type III (1) 60 Lesch-Nylan syndrome (3) Hyperphenylalanimemia, mild (3) ? Letterer-Siwe disease (2) Hyperproglucagonemia (1) Leukemia, acute lymphoblastic (1) Hyperproinsulinemia, familial (1) Leukemia, acute lymphoblastic (2) Hypertension, essential, 145500 (1) Leukemia, acute lymphocytic, with 4/11 Hypertension, essential, susceptibility to (3) translocation (3) Hypertriglyceridimia, one form (1) 65 Leukemia, acute myeloid (3) 21q22.3 Hypervalinemia or hyperleucine-isoleucinemia (1) Leukemia, acute myeloid, M2 type (1) Xp22.32 6,093,699 23 24

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Disorder Location Disorder Location Leukemia, acute pre-B-cell (2) Mental retardation, X-linked, syndromic-4, with Xq13-q22 Leukemia, acute promyelocytic (1) congenital contractures and low fingertip arches (2) Leukemia, acute promyelocytic (2) Mental retardation, X-linked, syndromic-5, with Leukemia, acute T-cell (2) Dandy-Walker malformation, basal ganglia disease, Leukemia, chronic myeloid (3) 22d 11.21 and seizures (2) Leukemia, chronic myeloid (3) Mental retardation, X-linked, syndromic-6, with Xp21.1-q22 Leukemia, multilineage (2) gynecomastia and obesity (2) Leukemia, myeloid/lymphoid of mixed-lineage (2) Mental retardation, X-linked-1, non-dysmorphic (2) Leukemia, T-cell acute lymphoblastic (2) Mental retardation, X-linked-2, non-dysmorphic (2) Leukemia, T-cell acute lymphoblastic (2) Mental retardation, X-linked-3 (2) Leukemia, T-cell acute lymphoblastoid (2) 3.2-p13.1 Mental retardation-skeletal dysplasia (2) Leukemia, T-cell acute lymphocytic (2) s Metahcromatic leukodystrophy (1) Leukemia, transient (2) 2 1.2 15 Metahcromatic leukodystrophy due to deficiency of Leukemia-1, T-cell acute lymphoblastic (3) SAF-1 (1) Leukemia-1, T-cell acute lymphoblastic (3) 9 E Methemoglobinemia due to cytochrome b5 deficiency (3) Chr.18 Leukemia/lymphoma, B-cell, 1 (2) 3.3 Methemoglobinemia due to enzymopathic (1) 22q13.31-qter Leukemia/lymphoma, B-cell, 2 (2) 21.3 Methemoglobinemias, alpha- (1) 6pter-p13.3 Leukemia/lymphoma, B-cell, 3 (2) Methemoglobinemias, beta- (1) 1p15.5 Leukemia/lymphoma, T-cell (2) 32.1 Methylmalonicaciduria, mutase deficiency type (1) Leukemia/lymphoma, T-cell (2) 2 Mevalonicaciduria (1) Chr. 12 Leukemia/lymphoma, T-cell (3) 1.2 Microphthalmia with linear skin defects (2) Xp22.2 Leukocyte adhesion deficiency (1) 22.3 Miller-Dieker lissencephaly syndrome (2) 7p13.3 Li-Fraumeni syndrome (1) 7 3.1 Mitochoedrial complex 1 deficiency, 252010 (1) Lipoamide dehydrogenase deficiency (1) -q32 MODY, one form (3) Lipoma (2) 2q13-q14 MODY, type I (2) Liver cell carcinoma (1) 1p14-p13 25 MODY, type II, 125851 (3) Long QT syndrome (2) Moebius syndrome (2) Lowe syndrome (3) Monocyte carboxyesterase deficiency (1) Lupus erythematosus, systemic, 152700 (1) Murolipidosis II (1) Lymphoproliferative syndrome, X-linked (2) Murolipidosis III (1) Lynch cancer family syndrome II (2) Mucopolysaccaridosis I (1) 2Lysosomal acid phosphatase deficiency (1) Mucopolysaccaridosis II (2) M Mucopolysaccaridosis IVA (3) Mucopolysaccaridosis IVB (1) Macrocytic anemia of 5q-syndrome, refractory (2) Mucopolysaccaridosis VII (1) Macrocrytic anemia refractory, of 5q-syndrome, Multiple endocrine neoplasia I (1) 153550 (3) Multiple endocrine neoplasia II (2) Macular dystrophy (1) 35 Multiple endocrine neoplasia III (2) Macular dystrophy, atypical vitelliform (2) Multiple exostoses (2) Macular dystrophy, North Carolina type (2) Multiple lipomatosis (2) Macular dystrophy, vitelliform type (2) Multiple scherosis, susceptibility to (2) Male infertility due to acrosin deficiency (2) Muscle glycogenosis (1) Male infertility, familial (1) Muscular dystrophy, Duchenne-like, autosomal (2) Male pseudohermaphroditism due to defective LH (1) Muscular dystrophy, limb-girdle, autosomal Malignant hyperthermia susceptibility-1, 145600 (3) 40 dominani (2) Malignant hyperthermia susceptibility-2, 145600 (2) Muscular dystrophy, limb-girdle, autosomal Malignant melanoma, cutaneous (2) recessive (2) 2Manic-depressive illness, X-linked (2) Myelodysplastic syndrome, preleukemic (3) Mannosidosis (1) Myelogenous leukemia, acute (3) Maple syrup urine disease, type 1 (3) Myeloperoxidase deficiency (1) Maple syrup urine disease, type 2 (3) 45 Myoadenylate deaminase deficiency (1) Maple syrup urine disease, type 3 (1) Myocardial infarction, susceptibility to (3) Marian syndrome, 154700 (3) Myoglobinuria/hemolysis due to PGK deficiency (1) Maroteraux-Lamy syndrome, several forms (1) Myopathy due to CTPase deficiency (1) Martin-Bell syndrome (2) Myopathy due to phosphoglycerate mutase MASA syndrome (2) deficiency (1) McArdle disease (1) 50 Myopia-1 (2) McCune-Albright polyostotic fibrous dysplasia, Myotonia congenita, atypical acetazolamide 174800 (1) responsive (2) McLeod phenotype (2) Myotonia congenita, dominant, 160800 (2) Medullary thyroid-carcinoma (2) Myotonia congenita, recessive, 255700 (3) Megacolon (2) Myotonic dystrophy (2) Megalocornea, X-linked (2) 55 Myotubular myopathy, X-linked (2) Melanoma, cutaneous malignant (2) 9p2 Myxoid liposarcoma (2) Meningioma (2) 22q12.3-qter N Meningioma (3) 22q12.3-q13.1 Menkes disease (2) Xq12-q13 2N syndrome, 310465 (1) Xp22.3-p21.1 Mental retardation of WAGR (2) 11p13 Nail-patella syndrome (2) 9q34 Mental retardation, Snyder-Robinson type (2) Xp21 Nance-Horan syndrome (2) Xp22.3-p21.1 Mental retardation, X-linked nonspecific, Xp11 60 Nemaline myopathy-1 (2) 1q21-q23 with aphasia (2) Nephromonnthsis, juvenile (2) 2p23-cen Mental retardation, X-linked, syndromic-1, with Xp22.2-p22.1 Neuroblastoma (2) 1p36.2-p36.1 dystonic movements, ataxia, and seizures (2) Neuroepithelioma, 133450 (1) 11q23-q24 Mental retardation, X-linked, syndromic-2, with Neuroepithelioma (2) 22q12 dysmorphism and cerebral atrophy (2) Neurofibromatosis, von Recklinghausen (3) 17q11.2 Mental retardation, X-linked, syndromic-3, with Xp11-q21.3 65 Neuropathy, recurrent, with pressure palsies, 17p11.2 spastic diplegia (2) 162500 (3) 6,093,699 25 26

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Disorder Location Disorder Location

Neutropenia, immune (2) 1q23 Porphyria, acute intermittent (1) C 24.1-q24.2 Niemann-Pick disease, type A (1) 11p15.4-15.1 Porphyria, Chester type (2) C Niemann-Pick disease, type B (1) 11p15.4-15.1 Porphyria, congenital erythropoietic (1) O q25.2-q26.3 Niemann-Pick disease, type C (2) 18p Porphyria cutanea tarde (1) 34 Nightblindness, congenital stationary, type 1 (2) Xp11.3 Porphyria, hepatoerythropoietic (1) 34 Non-insulin dependent diabetes aneltins, 19q13.3 Porphyria, variegata (2) d32 susceptibility to (2) Postanesthetic apnea (1) 26.1-q26.2 Norrie disease (2) Xp11.4 Prader-Willi syndrome (2) Norum disease (3) 16q22.1 Pre-eclampsia, susceptibility to (3) Nucleoside phosphorylase deficiency, 14q13.1 Progressive cone dystrophy (2) immunodeficiency due to (1) Prolidase deficiency (1) 9cen-q13.11 O Properdia deficiency, X-linked (3) Xp11.4-p11.23 15 Propionicacidemia, type I or pccA type (1) 3d32 Obesity (2) Propionicacidemia, type II or pccB type (1) 3q21-q22 Ocular albinism autosoma recessive (2) Protein C deficiency (1) 2q13-q14 Ocular albinis, Forsius-Erikson type (2) Protein C inhibitor deficiency (2) 4.d32.1 Ocular albinism, Nettleship-Falls type (2) Protein S deficiency (1) p11.1-11.2 Ornithine transcarbamylase deficiency (3) Proioporphyria, erythropoietic (1) 8pter-p11.21 Orofacial cleft (2) Pseudohermaphroditism, male, with gynecomastia (1) 7q11-q12 Oroticaciduria (1) Pseusohypoaldosteronism (1) d31.1 Osterarthrosis, precocious (3) Pseudohypoparathyroidism, type Ia (1) Od13.2 Osteogenesis imperfecta, 4 clinical forms, Pseudovaginal pertneocrotal, hypopadias (1) Chr.2 166200, 166210, 259420, 166220 (3) Pseudo-vitamin D dependency rickets I (2) 2a14 Osteogenesis imperfecta, 4 clinical forms, Pseudo-Zellweger syndrome (1) 3p23-p22 166200, 166210, 259420, 166220 (3) Pyrodoxine dependency with seizures (1) d31 Osteopetrosis, 257900 (1) 25 Pyropoikilocytosis (1) q21 Osteoporosis, idiopathic, 166710 (3) Pyruvate carboxylase deficiency (1) 1C Pyruvate dehydrogenase deficiency (1) Xp22.2-p22.1 Osteosarcoma, 25.9500 (2) Otopalatodigital syndrome, type I (2) Ovarian carcinoma, 16700 (2) Rabson-Mendenhall syndrome (1) Ovarian carcinoma (2) ?Ragweed sensitivity (2) Ovarian failure, premature (2) Reifenstein syndrome (1) Oxolosis (1) Renal cell carcinoma (2) Renal glucosuria (2) Renal tubular acidosis-Osteopetrosis syndrome (1) ?Paget disease of bone (2) 6p21.3 Retinal cone dystrophy-1 (2) ?Pallister-Hall syndrome (2) 3p25.3 35 Retinal cone-red dystrophy (2) Pancreatic lipase deficiency (1) 10q26.1 Retinis pigmentosa, autosomal dominant (1) ? Panhypopituitarism (1) 3q Retinitis pigmentosa, autosomal recessive (3) ? Panhypopituitarism, X-linked (2) Xq21.3-q22 Retinitis pigmentosa, peripheria-related (3) Paraganglioma (2) 11q22.3-q23.2 Retinitis pigmentosa-1 (2) Paramyuatonia congenita, 1683.00 (3) 17q23.1-q25.3 Retinitis pigmentosa-2 (2) Parathyroid adenomatosis 1 (2) 11q13 Retinitis pigmentosa-3 (2) ?Parietal formula (2) 11p12-p11.12 40 Retinitis pigmentosa-1, autosomal dominant (3) I?Parkinsonism, susceptibility to (1) 22q13.1 Retinitis pigmentosa-5 (2) Paraxysmal nocturnal hemoglobinuria (1) Xq22.1 Retinitis pigmentosa-6 (2) Pelizaeus-Merabacher disease (3) Retinitis pigmentosa-9 (2) Pelviareteric junction obstruction (2) Retinitis pigmentosa-10 (2) ?Pendered syndrome (2) Retinitis punctata albeceas (1) Periodintitis, juvenile (2) 4 q11-q13 45 Retinoblastoma (3) Persistent Mullerian duct syndrome (1) 9p13.3-p13.2 Retinol binding protein, deficiency of (1) Phenylketonuria (3) 2q24.1 Retinoschisis (2) Phenylketonuria due to dihydropteridine reductase 4p15.31 ?Rett syndrome (2) deficiency (1) Rhabdomyosarcoma (2) Pheochronocytoma (2) Rhabdomyosarcoma, alveolar (2) Phosphoribosyl pyrophosphatase synthetase 50 Rhabdomyosarcoma, alveolar (3) related gout (1) Ry-mull disease (1) 2Phospharylase kinase deficiency of liver and muscle, 2Rh-null hemolytic anemia (1) 261750 (2) Rickets, vitamin D-resistant (1) Piebaldism (3) Rieger syndrome (2) Pituitary tumro, growth-hormone-secreting (1) Rod monochromacy (2) PK deficiency hemolytic anemia (10 55 Rothmund-Thomas syndrome (2) Placental-lactogen deficiency (1) Rubinstein-Taybi syndrome (2) Placental steroid sulfatase deficiency (3) Russell-Silver syndrome (2) Plasmia inhibitor deficiency (1) S Plasminogen activator deficiency (1) Plasminogen deficiency, types I and II (1) Sacthre-Chotxen syndrome (2) Plasminogen Tochigi disease (1) Salivary gland pleomorphic adenoma (2) Platelet alphafdelta storage pool deficiency (1) 60 Sandhoff disease (1) Polio, susceptibility to (2) ?Sanfilippo disease, type IIIC (2) Polycystic kidney disease (2) Sanfilippo syndrome D (1) Sarcoma, synovial (2) Polycystic ovarian disease (1) Scheie syndrome (1) Polyposis coli, familial (3) ?Schizophrenia (2) Pompe disease (1) 65 Schizophrenia, chronic (3) Porphyria, acute hepatic (1) I?Schizophrenia, susceptibility to (2) 6,093,699 27 28

-continued -continued

Disorder Location Disorder Location Sclerotylosis (2) Usher syndrome, type 1C (2) 1p Severe combined immunodeficiency, 202500 (1) Usher syndrome, type 2 (2) q32 Severe combined immunodeficiency due to V ADA deficiency (1) Severe combined immunodeficiency due to van der Woude syndrome (2) q32 IL2 deficiency (1) Velocardiofacial syndrome (2) 22q11 Severe combined immunodeficiency, Vitreoretinopathy, exudative, familial (2) 1q13-q23 HLA class II-negative type (1) Vitreoretinopathy, neovascular inflammatory (2) 1q13 Severe combined immunodeficiency, X-linked, Vivax malaria, susceptibility to (1) q21-q22 300400 (3) von Hippel-Lindau syndrome (2) 3p26-p25 Short stature (2) von Willebrand disease (1) 2pter-p12 ?Sialidosis (2) Waardenburg syndrome, type I (3) 2a35 Sickle cell anemia (1) 15 Waardenburg syndrome, type III, 2a35 ?Simpson-Galabi-Behmel syndrome (2) Xcen-q22 48820 (3) Situs inversus vircerum (2) Waisman parkinsonism-mental retardation Xq28 SLE (1) syndrome (2) Small-cell cancer of lung (2) Watson syndrome, 193520 (3) 7q11.2 ?Smith-Lemli-Opitz syndrome (2) Werdnig-Hoffmann disease (2) 5q12.2-q13.3 Smith-Magenis syndrome (2) Werner syndrome (2) 8p12-p11 Spastic paraplegis, X-linked, uncomplicated (2) Wernicke-Korsakoff syndrome, susceptibility to 3p14.3 phereocytosis, hereditary (3) (1) pherocytosis, hereditary, Japanese type (1) Wieacher-Wolff syndrome (2) Xq13-q21 pherocytosis, recessive (1) Williams-Beuren syndrome (2) 4q33-q35.1 pherocytosis-1 (3) Wilms tumor (2) p13 pherocytosis-2 (3) Wilms tumor, type 2 (2) p15.5 pinal and bulbar muscular atrophy of Kennedy, 25 Wilson disease (2) 3q14-q21 13200 (3) Wiskott-Aldrich syndrome (2) Xp11.3-p11.2 pinal muscular atrophy II (2) Wolf-Hirschhorn syndrome, 194190 (3) 4p16.1 pinal muscular atrophy III (2) Wolf-Hirschhorn syndrome (2) 4p16.3 pinocerebellar ataxia-I (2) Wolman disease (1) Oq24-q25 pinocerebellar atrophy II (2) Wrinkly skin syndrome (2) 2C32 plit-hand/foot deformity, type 1 (2) X plit-hand/foot deformity, type 2 (2) pondyiopiphyseal dysplasia congenita (3) 2Xeroderma pigmentosum (1) q42 pondyiopiphyseal dysplasia tarda (2) Xeroderma pigmentosum, group B (3) 2d21 artle disease (2) Xeroderma pigmentosum, complementation Ch.5 icker syndrome (3) group C (2) Sucrose intolerance (1) 35 Xeroderma pigmentosum, group D, 278730 (1) 9q13.2-q13.3 Supravalvar aortic stenosis (3) Xeroderma pigmentosum, type A (1) 9q34.1 2Xeroderma pigmentosum, type F (2) Chr.15 Z. Tay-Sachs disease (1) Testicular feminizaiton (1) Zellweger syndrome, type II (1) 1p22-p21 Thalassemias, alpha- (1) 16pter-p13.3 Zellweger syndrome-1 (2) 7C11-23 Thalassemias, beta- (1) 11p15.5 40 Thrombocytopenia, X-linked (2) Xp21-p11 Thrombophilia due to elevated HRG (1) 3p14-qter (reprinted from Culver, K.W., “Gene Therapy”, 1994, p. 93, Thrombophilia due to excessive plasminogen activator 7q21.3-q22 Mary Ann Liebert, Inc., Publishers, New York, N.Y.). inhibitor (1) Alternatively tolerogenic conjugates may be Submitted in Thrombophilia due to heparin cofactor II deficiency (1) 45 accordance with U.S. Pat. No. 5,358,710 to Sehon et al., Thyroid hormone resistance, 264300, 188570 (3) incorporated herein by reference in its entirety. Thus the Thyroid iodine peroxidase deficiency (1) present invention can readily be adapted to any gene therapy Thyroid papillary carcinoma (1) protocol and is generally applicable to the administration of Thyrotropin-releaseing hormone deficiency (1) any therapeutic immunogenic material and not just the Torsion dystonia (2) Torsion dystonia-parkinsonism, Filipino type (2) 50 Specific examples listed above. Tourette syndrome (2) Gene therapy according to the existing art may be applied Transcobalamin II deficiency (1) to Somatic cells or germ line cells by methods known Such Transcortin deficiency (1) as gold electroporation, microinjection or jet injection, or Treacher Collins mandibulofacia dysoslosis (2) other methods as set forth in Sambrook et al. “Molecular Trichlorhinophalangeal syndrome, type I (2) Cloning: A Laboratory Manual, Cold Spring Harbor Press Trypsinogen deficiency (1) 55 I?Tuberculosis, susceptibility to (2) (1989)” incorporated herein by reference in its entirety. Tuberous sclerosis-1 (2) Thus the invention provides for a method for treating by Tuberous sclerosis-2 (2) gene therapy a mammal with a therapeutic amount of a Tuberous sclerosis-3 (2) biologically active antigenic material or its expression prod Tuberous sclerosis-4 (2) Turner syndrome (1) uct. To retain the effectiveness of Said antigenic material(s) Tyrostinemia, type I (1) 60 from counteraction by an antibody(ies) produced against Tyrostinemia, type II (1) it(them); it is essential to Suppress the capacity of the U recipient of the gene to mount an antibody response(s) to said biologically active antigenic material(s). This method Urate oxidase deficiency (1) 1p22 comprises: Urolithiasis, 2,8-dihydroxyadenine (1) 16q24 Usher syndrome, type 1A (2) 14q32 65 (a) Selecting a mammal which has not received prior Usher syndrome, type 1B (2) 11q13.5 exposure to Said biologically active antigenic material (S); 6,093,699 29 30 (b) administering to said mammal in Step (a) an immu immunogenic derivative thereof, wherein Said tolero noSuppressive effective amount of a tolerogenic cova genic conjugate of step (b) is administered at least one lent conjugate of Said biologically active antigenic day prior to said antigenic protein of Step (c). material, or an immunogenic fragment thereof, The purpose of the above description and examples is to covalently bound to monomethoxypolyethylene glycol illustrate Some embodiments of the present invention with of a molecular weight of about 4,500 to 10,000, out implying any limitation. It will be apparent to those of wherein Said poly(ethylene glycol) is monomethoxy skill in the art that various modifications and variations may polyethylene glycol and Said method Suppresses the be made to the composition and method of the present formation of about 98% of antibodies against said invention without departing from the underlying principles antigenic genetic material or its product, the effective or Scope of the invention. All patents and publications cited amount of Said conjugate Suppressing in Said mammal herein are incorporated by reference in their entireties. the formation of immunoglobulin antibodies against We claim: the antigenic genetic material(s), and Subsequently 1. In a method for gene therapy comprising administration (c) administering to said mammal atherapeutically effec of a gene therapy vector comprising a gene encoding an tive amount of Said biologically active antigenic 15 antigenic protein operably linked to a promoter, the genetic material alone, or a derivative thereof Synthe improvement comprising the Step of sized by conjugating to Said antigenic genetic material the DNA expressing biologically or pharmacologically at least one day prior to administration of Said gene active molecules, wherein Said tolerogenic conjugate of therapy vector, administering to a mammal a tolero Step (b) is administered at least one day prior to said genic conjugate consisting of a protein coupled to antigenic genetic material of Step (c). monomethoxypolyethylene glycol (mPEG) having a In an alternative embodiment, the invention provides a molecular weight of about 2,000-10,000 daltons, method for Suppressing the capacity of a mammal to wherein Said protein is identical to the antigenic protein mount an IgG class antibody response to a biologically encoded by Said gene, and wherein Said gene is 25 expressed at a level Sufficient for gene therapy as a active antigenic product of genetic material compris result of the Suppression of an immune response to the ing: antigenic protein encoded by Said gene. (a) Selecting a mammal which has not received prior 2. In a method for gene therapy according to claim 1, exposure to Said biologically active antigenic genetic wherein Said gene therapy vector is Selected from the group material; consisting of Moloney murine leukemia virus vector, aden (b) administering to said mammal of step (a), a tolero Ovirus vector, and vaccinia virus vector. genic covalent conjugate comprising Said biologically 3. In a method for gene therapy comprising administration active antigenic genetic material or an immunogenic of a DNA encoding an antigenic protein operably linked to fragment the re of, co Valently bound to a promoter, the improvement comprising the Step of monomethoxypoly(ethylene glycol) of a molecular 35 at least one day prior to administration of Said DNA, weight of about 4,500 to 10,000, in an immunosup administering to a mammal a tolerogenic conjugate pressive effective amount capable of Suppressing the consisting of a protein conjugated to monomethoxy formation of immunoglobulin antibodies of the IgG polyethylene glycol (mPEG) having a molecular immunoglobulin class against Said antigenic genetic weight of about 2,000-10,000 daltons, wherein said material; and Subsequently 40 protein is identical to the antigenic protein encoded by (c) administering to said mammal atherapeutically effec said DNA, and wherein said DNA is expressed at a tive amount of Said biologically active antigenic level Sufficient for gene therapy as a result of the genetic material alone or an immunogenic derivative of Suppression of an immune response to the antigenic Said genetic material, wherein Said mammal is Sup protein encoded by said DNA. pressed from mounting an IgG class antibody response 45 to Said biologically active antigenic genetic material or k k k k k