CA2123611A1 - Treatment of melanoma with antisense oligonucleotides to c-myb proto-oncogene - Google Patents
Treatment of melanoma with antisense oligonucleotides to c-myb proto-oncogeneInfo
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Abstract
Melanoma is treated by administering oligonucleotides having a nucleotide sequence complementary to at least a portion of the mRNA
transcript of the human c-myb gene. These "antisense"
oligonucleotides are hybridizable to the c-myb mRNA transcript.
transcript of the human c-myb gene. These "antisense"
oligonucleotides are hybridizable to the c-myb mRNA transcript.
Description
W093/09781) PCT/U~92t~9656 TREATMENT OF MELANOMA ~IT~
ANTI~ENBE O~IGONUCLEOTIDE~ TO C-MYB PROTO-ONCOGENE
Field of the Tnvention The invention relates to antisense oligonucleotides to proto-oncogenes, in particular antisense oligonucleotides to the c-mvb gene, and the use of such oligonucleotides as antineoplastic agents.
Reference to Government Grant The invention described herein was made in part with government support under grant CA 54384 awarded by National Institutes of Health. The government has certain rights in the invention.
Backaround of the Invention The proto-oncogene c-myb is the normal cellular homologue of the avian myeloblastosis virus-transforming gene v-myk. The c-mYb gene codes for a nuclear protein expressed primarily in hematopoietic cells. It is a proto-oncogene, that is, it codes for a protein which is required for the survival of normal, non-tumor oells.
When the gene is altered in the appropriate manner, it has the potential to become an oncogene. Oncogenes are genes whose expression within a cell provides some function in the transformation from normal to tumor cell.
The human c-myb gene ~as been isolated, cloned, and sequenced~ Majello et al., Proc. Natl. Acad. Sci.
U.S.A. 83, 9636-9640 (1986). Antisense oligonucleotides W O 93/09789 P ~ /US92/096~6
ANTI~ENBE O~IGONUCLEOTIDE~ TO C-MYB PROTO-ONCOGENE
Field of the Tnvention The invention relates to antisense oligonucleotides to proto-oncogenes, in particular antisense oligonucleotides to the c-mvb gene, and the use of such oligonucleotides as antineoplastic agents.
Reference to Government Grant The invention described herein was made in part with government support under grant CA 54384 awarded by National Institutes of Health. The government has certain rights in the invention.
Backaround of the Invention The proto-oncogene c-myb is the normal cellular homologue of the avian myeloblastosis virus-transforming gene v-myk. The c-mYb gene codes for a nuclear protein expressed primarily in hematopoietic cells. It is a proto-oncogene, that is, it codes for a protein which is required for the survival of normal, non-tumor oells.
When the gene is altered in the appropriate manner, it has the potential to become an oncogene. Oncogenes are genes whose expression within a cell provides some function in the transformation from normal to tumor cell.
The human c-myb gene ~as been isolated, cloned, and sequenced~ Majello et al., Proc. Natl. Acad. Sci.
U.S.A. 83, 9636-9640 (1986). Antisense oligonucleotides W O 93/09789 P ~ /US92/096~6
2 1 2 3 ~ 2 -to human c-myb mRNA that is, oligonucleotides having a nucleotide sequence complementary to the mRNA transcript of the c-m~b gene, are disclosed in our allowed, co-pending application Serial No. 427,659, filed October 27, 1989, and corresponding international patent application WO90/05445, the entire disclosures of which are incorporated herein by reference. C-myb antisense oligonucleotides are disclosed therein as being useful for the treatment of hematologic neoplasms, and for immunosuppression.
Melanoma, also known as "malignant melanoma"
or "cutaneous melanoma", is a neoplasm of melanocytes that has the potential for invasion and metastasis, Melanocytes are melanosome-containing cells that specialize in the biosynthesis and transport of melanin pigment. Melanocytes reside in the skin at the basal layer of the epidermis. Under a variety of stimuli, they elaborate melanin pigment. Melanin synthesis occurs on the melanosome, a well-defined intracellular organelle within the melanosome.
At one time considered rare, the rate of increase in the incidence of melanoma i5 greater than for any other cancer, with the exception of bronchogenic carcinoma. The incidence of melanoma is greatest among Caucasians, and is influenced by ultraviolet light exposure, and by geographical and occupational factors.
~he incidence of melanoma is increasing rapidly in the United States and elsewhere, with an apparent doubling every ten to seventeen years. Presently, melanoma accounts for roughly one percent of cancers in the United States, and about the same proportion of cancer deaths.
While it represents only about three percent of cutaneous neoplasms, melanoma accounts for two thirds of all skin cancer fatalities.
For the most part, melanoma first progr~s~s through a radial growth phase at the site of the primary lesion. This initial phase is characterized by little ~'l23~
wo93/o978s PCT/US92/09656 or no competence to metastasize. Melanomas in this phase are generally treatable by surgical procedures. In the vertical growth phase, characterized by penetration into deeper cutaneous tissues, a primary melanoma acquires competence to metastasize. Surgery alone is ineffective in treating the melanoma, once metastasis has occurred.
Chemotherapy, either alone or in combination with surgery, has been utili~ed in the treatment of melanoma. Dimethyltriazeno-imidazole (DTIC) is the most active single agent for the treatment of metastatic melanoma, with an overall objective response rate of only 21%. DTIC can cause disturbances in liver function. It possesses modest hematologic toxicity.
Somewhat less effective in treating malignant melanoma are the synthetic nitrosoureas, of which 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrourea tCCNU), methyl-CCNU
and chlorozotocin are best known. The nitrosoureas display a somewhat more severe hematologic toxicity than DTIC. Response rates range from 10% to 18%.
Unfortunately, there are no single agents or combination regimens that induce a substantial number of complete remissions in melanoma patients. There is an urgent need for the development of more effective agents.5 summarY of the Invention The invention provides a method for treating melanoma. An effective amount of one or more c-myb antisense oligonucleotides is administered to an individual in need of such treatment. Each oligonucleotide has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-mvb gene. The oligonucleotide is hybridizable to the mRNA transcript. Preferably, the oligonucleotide is at least a 12-mer oligonucleotide, that is, an oligomer containing at least 12 nucleotide residues. In particular, the oligomer is advantageou91y a 12-mer to W093/09789 PCT/US92~09656 21~611 4 a 40-mer, preferably an oligodeoxynucleatide. While oligonucleotides smaller than 12-mers may be utilized, they are statistically more likely to hybridize with non-targeted sequences, and for this reason may be less specific. In addition, a single mismatch may destabilize the hybrid. While oligonucleotides larger than 40-mers may be utilized, uptake may be more difficult. Moreover, partial matching of long sequences may lead to non-specific hybridization, and non-specific effects.
Preferably, the oligonucleotide is a 15- to 3~-mer oligodeoxynucleotide, more advantageously an 18- to 26-mer.
While in principle oligonucleotides having a sequence complementary to any region of the c-~y~ gene find utility in the present invention, oligodeoxynucleo-tides complementary to a portion of the c-myb mRNA trans-cript including the translation initiation codon are particularly preferred. Also preferred are oligonucleotides complementary to a portian of the c-myb ~20 mRNA transcript lying within about 40 nucleotides ; upstream (the 5' direction) or about 40 nucleotides down-stream (the 3' direction) from the translation initiation codon.
The invention is also a method for purging bone marrow of metastasized melanoma cells. Bone marrow aspirated from a melanoma-inflicted individual is treated with an effective amount of c-~y~ antisense oligonucleotide, and the thus-treated cells are then returned to the body of the afflicted individual. The bone marrow purging technique may be utilized for an autologous bone marrow rescue (transplantation), in connection with a course of high dose chemotherapy.
The inYention is also a composition for the treatment of melanoma comprising a pbarmaceutically acceptable carrier and c-myb antisense oligonuclootide.
As used in the herein specification and appended claims, unless otherwise indicated, the term W O 93/09789 PC~/US92/09656 s ?l23~ll "oligonucleotide" includes both oligomers of ribonucleotides, i.e., oligoribonucleotides, and oligomers of deoxyribonucleotides, i.e., oligo-deoxyribonucleotides ~also re~erred to herein as "oligo-deoxynucleotides"). Oligodeoxynucleotidesarepreferred.
As used herein, unless otherwise indicated, the term "oligonucleotide" also includes oligomers which may be large enough to be termed "polynucleotides".
The terms"oligonucleotide"and"oligodeoxynuc-leotide" include not only oligomers and polymers of thecommon biologically significant nucleotides, i.e., the nucleotides adenine ("A"), deoxyadenine ("dA"), guanine ("G"), deoxyguanine ("dG"), cytosine ("C"), deoxycytosine . ("dC"), thymine ("T") and uracil ("U"), but also include oligomers and polymers hybridizable to the c-myb mRNA
transcript which may contain other nucleotides.
Likewise, the terms "oligonucleotide" and "oligodeoxynucleotide" includes oligomers and polymers wherein one or more purine or pyrimidine moieties, sugar moieties or internucleotide linkages is chemically modified. The term "oligonucleotide" is thus understood to also include oligomers which may properly be designated as "oligonucleosides" because of modification of the internucleotide phosphodiester bond. Such modified oligonucleotides include, for example, the methylphosphonate oligonucleosides, discussed below.
The term "phosphorothioate oligonucleotide"
means an oligonucleotide wherein one or more of the internucleotide linkages is a phosphorothioate group, --O -- P -- O~
W O 93/09789 PC~r/US92/09656 212~ 6 _ as opposed to the phosphodiester group o ~o - P - 0~
which is characteristic of unmodified oligonucleotides.
By "methylphosphorate oligonucleoside" ismeant an oligonuclèotide wherein one or more of the internucleotide linkages s a methylphosphonate group, ll --O -- P -- O--The term "downstream" when used in reference to a direction along a nucleotide sequence means the 5'~3' direction. Similarly, the term "upstream" means the 3'~5' direction.
The term "c-mYb mRNA transcript" means the presently known mRNA transcript of the human c-mvb gene, or any further transcripts which may be elucidated.
Brief Des¢ri~tion of the F~oures Fig. 1 is a graph of the effect of c-myb sense and antisense oligonucleotide on human melanoma cells (CHP) n vitro. Cells were treated with ~i) no oligomer ("Control"), (ii) the indicated concentrations of an 18-mer oligodeoxynucleotide complementary to codons 2-? of the translated position of the c-~y~ mRNA transcript ("Antisense"), or (iii) the corresponding sense 18-mer ("Sense").
Fig. 2 is similar to Fig. 1, except that the oligonucleotide treatment was extended for two days. The oligonucleotide concentrations are cumulative of the two day treatment period.
' ~093/09789 2 1 2 3 6 1 lPCT/~JS92/09656 Fig. 3 is similar to Fig. 2, except that the treatment was extended to five consecutive days. The oligonucleotide concentrations are cumulative.
Fig. 4 is similar to Fig. 1, except that another human melanoma cell line (SK MEL-37) was substituted for the CHP cells.
Fig. 5 is a plot of the effect of c-myb sense and antisense oligomers on the growth of human melanoma ~SK MEL-37) cells transplanted into severe combined immunodeficient mice. The mice received 100 ~g per day for 7 days of (i) a 24-mer phosphorothioate oligodeoxynucleotide complementary to codons 2-9 of the translated portion of the c-myb mRNA transcript ("Antisense"; 2 mice), (ii) the corresponding sense 24-lS mer phosphorothioate oligodeoxynucleotide ("Sense"; 1mouse) or ~iii) no oligomer ("Control"; 1 mouse).
Detailea De~criDtion of the Invention It has now been discovered that the expression of the human c-myb gene is important for the proliferation of malignant melanoma. The role of this proto-oncogene in cell proliferation is not restricted to cells of hematopoietic ori~in, as previously thought.
The proliferation of malignant melanocytes, which are neoplastic cells of epidermal and not hematologic origin, is maintained by c-myb expression. Thus, the role of c-mYb is more general than previously thought.
The putative DNA sequence complementary to the mRNA transcript of the human c-~y~ gene has been reported in Majello et al., Proc. Natl. Acad. Sci. U.S.A. 83, 9636-9640 (1986). Majello et ~1. further disclose the predicted 640 amino acid sequence of the putative c-my~
protein. The initiation codon ATG appears at position 114, preceded by a 5'-untranslated region. The termination codon TGA at position 2034 is followed by a
Melanoma, also known as "malignant melanoma"
or "cutaneous melanoma", is a neoplasm of melanocytes that has the potential for invasion and metastasis, Melanocytes are melanosome-containing cells that specialize in the biosynthesis and transport of melanin pigment. Melanocytes reside in the skin at the basal layer of the epidermis. Under a variety of stimuli, they elaborate melanin pigment. Melanin synthesis occurs on the melanosome, a well-defined intracellular organelle within the melanosome.
At one time considered rare, the rate of increase in the incidence of melanoma i5 greater than for any other cancer, with the exception of bronchogenic carcinoma. The incidence of melanoma is greatest among Caucasians, and is influenced by ultraviolet light exposure, and by geographical and occupational factors.
~he incidence of melanoma is increasing rapidly in the United States and elsewhere, with an apparent doubling every ten to seventeen years. Presently, melanoma accounts for roughly one percent of cancers in the United States, and about the same proportion of cancer deaths.
While it represents only about three percent of cutaneous neoplasms, melanoma accounts for two thirds of all skin cancer fatalities.
For the most part, melanoma first progr~s~s through a radial growth phase at the site of the primary lesion. This initial phase is characterized by little ~'l23~
wo93/o978s PCT/US92/09656 or no competence to metastasize. Melanomas in this phase are generally treatable by surgical procedures. In the vertical growth phase, characterized by penetration into deeper cutaneous tissues, a primary melanoma acquires competence to metastasize. Surgery alone is ineffective in treating the melanoma, once metastasis has occurred.
Chemotherapy, either alone or in combination with surgery, has been utili~ed in the treatment of melanoma. Dimethyltriazeno-imidazole (DTIC) is the most active single agent for the treatment of metastatic melanoma, with an overall objective response rate of only 21%. DTIC can cause disturbances in liver function. It possesses modest hematologic toxicity.
Somewhat less effective in treating malignant melanoma are the synthetic nitrosoureas, of which 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrourea tCCNU), methyl-CCNU
and chlorozotocin are best known. The nitrosoureas display a somewhat more severe hematologic toxicity than DTIC. Response rates range from 10% to 18%.
Unfortunately, there are no single agents or combination regimens that induce a substantial number of complete remissions in melanoma patients. There is an urgent need for the development of more effective agents.5 summarY of the Invention The invention provides a method for treating melanoma. An effective amount of one or more c-myb antisense oligonucleotides is administered to an individual in need of such treatment. Each oligonucleotide has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-mvb gene. The oligonucleotide is hybridizable to the mRNA transcript. Preferably, the oligonucleotide is at least a 12-mer oligonucleotide, that is, an oligomer containing at least 12 nucleotide residues. In particular, the oligomer is advantageou91y a 12-mer to W093/09789 PCT/US92~09656 21~611 4 a 40-mer, preferably an oligodeoxynucleatide. While oligonucleotides smaller than 12-mers may be utilized, they are statistically more likely to hybridize with non-targeted sequences, and for this reason may be less specific. In addition, a single mismatch may destabilize the hybrid. While oligonucleotides larger than 40-mers may be utilized, uptake may be more difficult. Moreover, partial matching of long sequences may lead to non-specific hybridization, and non-specific effects.
Preferably, the oligonucleotide is a 15- to 3~-mer oligodeoxynucleotide, more advantageously an 18- to 26-mer.
While in principle oligonucleotides having a sequence complementary to any region of the c-~y~ gene find utility in the present invention, oligodeoxynucleo-tides complementary to a portion of the c-myb mRNA trans-cript including the translation initiation codon are particularly preferred. Also preferred are oligonucleotides complementary to a portian of the c-myb ~20 mRNA transcript lying within about 40 nucleotides ; upstream (the 5' direction) or about 40 nucleotides down-stream (the 3' direction) from the translation initiation codon.
The invention is also a method for purging bone marrow of metastasized melanoma cells. Bone marrow aspirated from a melanoma-inflicted individual is treated with an effective amount of c-~y~ antisense oligonucleotide, and the thus-treated cells are then returned to the body of the afflicted individual. The bone marrow purging technique may be utilized for an autologous bone marrow rescue (transplantation), in connection with a course of high dose chemotherapy.
The inYention is also a composition for the treatment of melanoma comprising a pbarmaceutically acceptable carrier and c-myb antisense oligonuclootide.
As used in the herein specification and appended claims, unless otherwise indicated, the term W O 93/09789 PC~/US92/09656 s ?l23~ll "oligonucleotide" includes both oligomers of ribonucleotides, i.e., oligoribonucleotides, and oligomers of deoxyribonucleotides, i.e., oligo-deoxyribonucleotides ~also re~erred to herein as "oligo-deoxynucleotides"). Oligodeoxynucleotidesarepreferred.
As used herein, unless otherwise indicated, the term "oligonucleotide" also includes oligomers which may be large enough to be termed "polynucleotides".
The terms"oligonucleotide"and"oligodeoxynuc-leotide" include not only oligomers and polymers of thecommon biologically significant nucleotides, i.e., the nucleotides adenine ("A"), deoxyadenine ("dA"), guanine ("G"), deoxyguanine ("dG"), cytosine ("C"), deoxycytosine . ("dC"), thymine ("T") and uracil ("U"), but also include oligomers and polymers hybridizable to the c-myb mRNA
transcript which may contain other nucleotides.
Likewise, the terms "oligonucleotide" and "oligodeoxynucleotide" includes oligomers and polymers wherein one or more purine or pyrimidine moieties, sugar moieties or internucleotide linkages is chemically modified. The term "oligonucleotide" is thus understood to also include oligomers which may properly be designated as "oligonucleosides" because of modification of the internucleotide phosphodiester bond. Such modified oligonucleotides include, for example, the methylphosphonate oligonucleosides, discussed below.
The term "phosphorothioate oligonucleotide"
means an oligonucleotide wherein one or more of the internucleotide linkages is a phosphorothioate group, --O -- P -- O~
W O 93/09789 PC~r/US92/09656 212~ 6 _ as opposed to the phosphodiester group o ~o - P - 0~
which is characteristic of unmodified oligonucleotides.
By "methylphosphorate oligonucleoside" ismeant an oligonuclèotide wherein one or more of the internucleotide linkages s a methylphosphonate group, ll --O -- P -- O--The term "downstream" when used in reference to a direction along a nucleotide sequence means the 5'~3' direction. Similarly, the term "upstream" means the 3'~5' direction.
The term "c-mYb mRNA transcript" means the presently known mRNA transcript of the human c-mvb gene, or any further transcripts which may be elucidated.
Brief Des¢ri~tion of the F~oures Fig. 1 is a graph of the effect of c-myb sense and antisense oligonucleotide on human melanoma cells (CHP) n vitro. Cells were treated with ~i) no oligomer ("Control"), (ii) the indicated concentrations of an 18-mer oligodeoxynucleotide complementary to codons 2-? of the translated position of the c-~y~ mRNA transcript ("Antisense"), or (iii) the corresponding sense 18-mer ("Sense").
Fig. 2 is similar to Fig. 1, except that the oligonucleotide treatment was extended for two days. The oligonucleotide concentrations are cumulative of the two day treatment period.
' ~093/09789 2 1 2 3 6 1 lPCT/~JS92/09656 Fig. 3 is similar to Fig. 2, except that the treatment was extended to five consecutive days. The oligonucleotide concentrations are cumulative.
Fig. 4 is similar to Fig. 1, except that another human melanoma cell line (SK MEL-37) was substituted for the CHP cells.
Fig. 5 is a plot of the effect of c-myb sense and antisense oligomers on the growth of human melanoma ~SK MEL-37) cells transplanted into severe combined immunodeficient mice. The mice received 100 ~g per day for 7 days of (i) a 24-mer phosphorothioate oligodeoxynucleotide complementary to codons 2-9 of the translated portion of the c-myb mRNA transcript ("Antisense"; 2 mice), (ii) the corresponding sense 24-lS mer phosphorothioate oligodeoxynucleotide ("Sense"; 1mouse) or ~iii) no oligomer ("Control"; 1 mouse).
Detailea De~criDtion of the Invention It has now been discovered that the expression of the human c-myb gene is important for the proliferation of malignant melanoma. The role of this proto-oncogene in cell proliferation is not restricted to cells of hematopoietic ori~in, as previously thought.
The proliferation of malignant melanocytes, which are neoplastic cells of epidermal and not hematologic origin, is maintained by c-myb expression. Thus, the role of c-mYb is more general than previously thought.
The putative DNA sequence complementary to the mRNA transcript of the human c-~y~ gene has been reported in Majello et al., Proc. Natl. Acad. Sci. U.S.A. 83, 9636-9640 (1986). Majello et ~1. further disclose the predicted 640 amino acid sequence of the putative c-my~
protein. The initiation codon ATG appears at position 114, preceded by a 5'-untranslated region. The termination codon TGA at position 2034 is followed by a
3'-untranslated region spanning about 1200 nucleotides, WO 93/09789 PCI/US92/096~6 212361~ - 8 - ~
which is followed by a poly (A) tail of about 14 0 nucleotides.
The antisense oligonucleotides of the invention may be synthesized by any of the known chemical oligonu-cleotide synthesis methods. Such methods are generallydescribed, for example, in Winnacker, From Genes to Clones: Introduction to Gene Technoloqv, VCH Verlagsges-ellschaft mbH (Ibelgaufts trans. 1987). The antisense oligonucleotides are most advantageously prepared by utilizing any of the commercially available, automated nucleic acid synthesizers. One such device, the Applied ~-Biosystems 380B DNA Synthesizer, utilizes ~-cyanoethyl phosphoramidite chemistry.
Since the complete nucleotide synthesis of DNA
complementary to the c-myb mRNA transcript is known, an-tisense oligonucleotides hybridizable with any portion of the mRNA transcript may be prepared by oligonucleotide synthesis methods known to those skilled in the art.
While any length oliqonucleotide may be utilized in the practice of the invention, sequences shorter than 12 bases may be less specific in hybridizing to the target c-mvb mRNA, may be more easily destroyed by enzymatic digestion, and may be destabilized by enzymatic digestion. Hence, oligonucleotides having 12 or more nucleotides are preferred.
Long sequences, particularly sequences longer than about 40 nucleotides, may be somewhat less effective in inhibiting c-myb translation because of decreased uptake by the target cell. Thus, oligomers of 12-40 nucleotides are preferred, more preferably 15-30 nucleotides, most preferably 18-26 nucleotides. While sequences of 18-21 nucleotides are most particularly preferred, for unmodified oligonucleotides, slightly longer chains of up to about 26 nucleotides, are preferred for modified oligonucleot~des such as phosphorothioate oligonucleotides, which hybridize less strongly to mRNA than unmodified oligonucleotides. `
. .
9 ~12~61~
Oligonucleotides complementary to and hybridizable with any portion of the c-myb mRNA
transcript are, in principle, effective for inhibiting translation of the transcript, and capable of inducing the effects herein described. It is believed that translation is most effectively inhibited by blocking the mRNA at a site at or near the initiation codon. Thus, oligonucleotides complementary to the 5'-terminal region of the c-mYb mRNA transcript are preferred. It is believed that secondary or tertiary structure which might interfere with hybridization is minimal in this region.
Moreover, it has been suggested that sequences that are too distant in the 3'-direction from the initiation site may be less effective in hybridizing the mRNA transcripts because of a "read-through" phenomenon whereby the ribosome is postulated to unravel the antisense/sense duplex to permit translation of the message. See, e.g., Shakin, J. Biochemistry 261, 16018 tl986).
The antisense oligonucleotide is preferably directed to a site at or near the initiation codon for protein synthesis. Oligonucleotides complementary to the c-my~ mRNA, including the initiation codon (the first codon at the 5' end of the translated portion of the c-EY~ transcript, comprising nucleotides 114-116 of the complete transcript) are preferred.
While antisense oligomers complementary to the ~'-terminal region of the c-mYb transcript are preferred, particularly the region including the initiation codon, it should be appreciated that usaful antisense oligomers are not limited to those complementary to the sequenceæ
found in the translated portion (nucleotides 114 to 2031) of the mRNA transcript, but also includes oligomers complementary to nucleotide sequences contained in, or exténding into, the 5'-and 3'-untranslated regions.
Oligomers whose complementarity extends into the 5'-untranslated region o~ the c-myb transcript are believed particularly effective in inhibiting c-_y~ translation.
212~611 10 Preferred oligonucleotidescomplementary tot~e 5'-untranslated region of the transcript include molecules having a nucleotide sequence complementary to a portion of the c-mvb mRNA transcript including the cap nucleotide, that is, the nucleotide at the extreme 5'-end of the transcript. The Sl nuclease assay procedure of Molecular Clonina, 2nd edition (Sambrook et al., Eds.
1989), pages 7.66-7.70 (incorporated herein by reference) was essentially followed to map the location of c-~yk cap sites using mRNA isolated from the leukemic cell line CCRF-CEM, which expresses high levels of c-~y~ mRNA. The longest clearly visible band was located 90 base pairs upstream of the published c-mvb cDNA (Majello ç~ al., Proc. ~atl. Acad. Sci. U.S.A., 83, 9536-9640 (1986)), indicating the putative principle cap site. The position of this site is in perfect agreement with the length of the c-myb cDNA cloned from CCRF-CEN cells (Clarke et al., Mol. Cell. Biol., 8, 884-892 (1988)). Sl protection ~; assays also revealed faint bands in addition to the main band corresponding to the cap site. These other bands may represent rare or unstable c-Ey~ mRNA transcripts.
Multiple sites of transcription initiation are not uncommon in genes such as c-myb which lack a perfect TATAA box. The nucleotide sequence of the mRNA
transcript 5'-terminus beginning with the cap nucleotide may be readily established, and antisense oligonuc-leotides complementary and hybridizable thereto may be prepared.
The following 40-mer oligodeoxynucleotide is complementary to the c-mYb mRNA transcript beginning with the initiation codon of the transcript and extending downstream thereof (in the 3' direction): SEQ ID N0:1.
Smaller oligomers based upon the above sequence, in particular, oligomers hybridizable to segments of the c-mvb message containing the initiation codon, may be utilized. Particularly pr~ferrQd are the following 26- to 15-mers: ~
.
W0~3/09789 PCT/US92~09656 1l 2123611 SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ I~ NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SE~ ID N0:12 and SEQ ID NO:13.
Oligodeoxynucleotides complementary to the c-myb mRNA transcript beginning with the second codon of the translated portion of the transcript (nucleotides 117-119 of the complete transcript) are another group of preferred oligomers. Such oligomers include, for example, the following 26- to 15-mers:
SEQ ID N0:14, SE~ ID N0:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23, 3Q SEQ ID N0:24 and SEQ ID N0:25.
The oligonucleotide employed may represent an un-modified or modified oligonucleotide. Thus, oligo-nucleotides hybridizable to the c-~y~ mRNA transcript finding utility according to the present invention include not only oligomers of the biologically sig-W O 93/09789 P ~ tUS92/09656 2 1 ~ 12 -nificant native nucleotides, i.e., A, dA, G, dG, C, dC, T and U, but also oligonucleotide species which have been modified for improved stability and/or lipid solubility~
For example, it i5 known that enhanced lipid solubility and/or resistance to nuclease digestion results by substituting an alkyl group or alkoxy group for a phos-phate oxygen in the internucleotide phosphodiester linkage to form an alkylphosphonate oligonucleoside or alkylphosphotriester oligonucleotide. Non-ionic oligonucleotides such as these are characterized by increased resistance to nuclease hydrolysis and/or increased cellular uptake, while retaining the ability to form stable complexes with complementary nucleic acid sequences. The alkylphosphonates in particular, are stable to nuclease cleavage and soluble in lipid. The preparation of alkylphosphonate oligonucleosides is disclosed in U.S. Patent 4,469,863.
Methylphosphonate oligomers can be prepared by a variety of methods, both in solution and on insoluble polymer supports (Agrawal and Riftina, Nucl. Acids Res., 6, 3009-3024 (1979); Miller et al., Biochemistry, 18, 5134-5142 (1979), Miller et al., J. Biol. Chem., 255, 9659-9665 (1980); Miller et al., Nucl. Acids Res., 11, 5189-5204 (1983), Miller et al., Nucl. Acids Res., 11, 6225-6242 (1983), Miller et al., Biochemistry, 25, 5092-5097 (1986); Engels and Jager, Anqew. Chem. Suppl. 912 (1982); Sinha et al., Tetrahedron Lett. 24, 877-880 (1983); Dorman et al, Tetrahedron, 40, 95~102 tl984);
Jager and Engels, Tetrahedron Lett., 25, 1437-1440 (1984); Noble et al., Nucl. Acids Res., 12, 3387-3404 ~1984); Callahan et al., Proc. Natl. Acad. Sci. USA, 83, 1617-1621 (1986); Koziolkiewicz e~ hemica Scri~ta, 26, 251-260 (1986): Agrawal and Goodchild, Tetrahedron Lett., 38, 3539-3542 (1987); Lesnikowski et ~1-.
Tetrahedron Lett., 28, 5535-5538 (1987); Sarin et al., Proc. Natl. Acad. Sci. USA, 85~ 7448-7451 ~1988)).
The most efficient procedure for preparation of methylphosphonate oligonucleosides involves use of 5'-o-d i m e t h o x y t r i t y l d e o x y n u c l e o s i d e - 3 ' - Q-diisopropylmethylphosphoramidite intermediates, which are similar to the methoxy or ~-cyanoethyl phosphoramidite reagents used to prepare oligodeoxyribonucleotides. The methylphosphonate oligomers can be prepared on controlled pore glass polymer supports using anautomated DNA
synthesizer (Sarin et al., Proc. Natl. Acad. Sci. USA, 85, 7448-7451 (1988)).
Resistance to nuclease digestion may also be achieved by modifying the internucleotide linkage at both the 5' and 3' termini with phosphoroamidites according to the procedure of Dagle et al., Nucl. Acids Res. 18, 4751-4757 (1990).
Phosphorothioate oligonucleotides contain a sulfur-for-oxygen substitution in the internucleotide phosphodiester bond. Phosphorothioate oligonucleotides combine the properties of effective hybridization for duplex formation with substantial nuclease resistance, while retaining the water solubility of a charged phosphate analogue. The charge is believed to confer the property of cellular uptake via a receptor (Loke et al., Proc. Natl. Acad Sci. U.S.A. 86, 3474-3478 (1989)).
Phosphorothioate oligodeoxynucleotide are described by LaPlanche, et al., Nucleic Acids Research 14, 9081 (1986) and by Stec et al., J. Am. Chem. Soc. 106, 6077 (lg84). The general synthetic method for phosphorothio-ate oligonucleotides was modified by Stein et al., Nucl.
Acids Res., 16, 3209-3221 (1988), so that these compounds may readily be synthesized on an automatic synthesizer using the phosphoramidite approach.
Furthermore, recent advances in the production of oligori~onucleotide analogues mean that other agents may also be used for the purposes described here, e.g., 2'-0-methylribonucleotides (Inove et al., ~ucleic Acids Res.
15, 6131 (1987) and chimeric oligonucleotides th,at are W O 93/09789 PC~r/US92/09656 ~ 3 6 1 ~ 14 _ composite RNA-DNA analogues (Inove et al., FEss Lett.
215, 327 (1987).
While inhibition of c-mvb mRNA translation is possible utilizing either antisense oligoribonucleotides or oligodeoxyribonucleotides, free oligoribonucleotides are more susceptible to enzymatic attack by ribonucleases than oligodeoxyribonucleotides. Hence, oligodeoxyribonucleotides are preferred in the practice of the present invention. Oligodeoxyribonucleotides are further preferred because, upon hybridization with c-myb mRNA, the resulting DNA-RNA hybrid duplex is a substrate for RNase H, which specifically attacks the RNA portion of DNA-RNA hybrid. Degradation of the mRNA strand of the duplex releases the antisense oligodeoxynucleotide strand for hybridization with additional c-myb messages.
In general, the antisense oligonucleotides used in the method of the present invention will have a sequence which is completely complementary to the target portion of the c-myb message. Absolute complementarity is not however required, particularly in larger oligomers.
Thus, reference herein to a "nucleotide sequence complementary to at least a portion of the mRNA
transcript" of c-mYb does not necessarily mean a sequence having 100% complementarity with the transcript. In gene-ral, any oligonucleotide having sufficient com-plementarity to form a stable duplex with c-mYb mRNA is suitable. Stable duplex formation depends on the sequence and length of the hybridizing oligonucleotide and the degree of complementarity with the target region of the c-myb message. Generally, the larger the hybridizing oligomer, the more mismatches may be tolerated. More than one mismatch probably will not be tolerated for antisense oligomers of less than about 21 nucleotides. One skilled in the art may readily determine the degree of mismatching which may be tolerated between any given antisense oligomer and the target c-mYb message sequence, based upon the melting wo93/o978s PCT/US92/09656 point, and therefore the stability, of the resulti~g duplex. Melting points of duplexes of a given base pair composition can be readily determined from standard texts, such as Molecular Cloninq: A Laboratory Manual, (2nd edition, 1989), J. Sambrook et al., eds.
While oligonucleotides capable of stable hybridiza-tion with any region of the c-myb message are within the scope of the present invention, oligonucleotides complementary to a region including the initiation codon are believed particularly effective. Particularly preferred are oligonucleotides hybridizable to a region of the c-~y~ mRNA up to 40 nucleotides upstream (in the 5' direction) of the initiation codon or up to 40 nucleotides downstream (in the 3' direction) of that codon.
For therapeutic use, the antisense oligonucleotides may be combined with a pharmaceutical carrier, such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives. The liguid vehicles and excipients are conventional and commercially available.
Illustrative thereof are distilled water, physiological saline, aqueous solution of dextrose, and the like. The c-~yb mRNA antisense oligonucleotides are preferably administeredparenterally,mostpreferably intravenously.
The vehicle is designed accordingly. Alternatively, oligonucleotide may be administered subcutaneously via controlled release dosage forms.
The oligonucleotides may be conjugated to poly(L-lysime) to increase cell penetration. Such conjugates are described by Lemaitre et al., Proc. Natl. Acad. 5ci~
PSA, 84, 648-652 (1987). The procedure requires that the 3'-terminal nucleotide be a ribonucleotide. The resulting aldehyde groups are then randomly coupled to the epsilon-amino groups of lysine residues of poly(L-lysine) by Schiff base formation, and then reduced withsodium cyanoborohydride. This procedure converts the 3'-terminal ribose ring into a morpholine structure antisense oligomers.
In addition to administration with conventional carriers, the antisense oligonucleotides may be administered by a variety of specialized oligonucleotide delivery techniques. For example, oligonucleotides may be encapsulated in for therapeutic delivery. The oligonucleotide, depending upon its solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspension. The hydrophobic layer, generally but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, ionic surfactants such as diacetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature. Oligonucleotides have been successfully encapsulated in unilameller liposomes.
Reconstituted Sendai virus envelopes have been suc-cessfully used to deliver RNA and DNA to cells. Arad et al., Biochem. Biophy. Acta. 859, 88-94 (1986).
The c-mvb antisense oligonucleotides may be administered de novo as the primary therapy.
Alternatively, the oligonucleotides may be administered as an adjuvant following surgical removal of a melanoma to patients who may be disease-free but at high risk of recurrence.
A preferred method of administration of oligonucleotide for treatment of melanoma comprises either regional or systemic perfusion, as is appropriate.
According to a method of regional perfusion, the afferent and efferent vessels supplying the extremity containing the lesion are isolated and connected to a low-flow perfusion pump in continuity with ~n oxygenator and a heat exchanger. The iliac vessels may be used for perfusion of the lower extremity. The axillary vessels are cannulated high in the axilla for upper extremity lesions. Oligonucleotide is added to the perfusion W093/09789 2 1 2 ~ PCT/US92/09656 circuit, and the perfusion is continued for an appropriate time period, e.g., one hour. Perfusion rates of from 100 to 150 ml/minute may be employed for lower extremity lesions, while half that rate should be employed for upper extremity lesions. Systemic heparinization may be used throughout the perfusion, and reversed after the perfusion is complete. This isolation perfusion technique permits administration of higher doses of chemotherapeutic agent than would otherwise be tolerated upon infusion into the arterial or venous sys-temic circulation.
For systemic infusion, the oligonucleotides are preferably delivered via a central venous catheter, which is connected to an appropriate continuous infusion device. Indwelling catheters provide long term access to the intravenous circulation for freguent administration of drugs over extended time periods. They are generally surgically inserted into the external cephalic or internal jugular vein under general or local anesthesia. The subclavian vein is another common site of catheterization. The infuser pump mày be external, or may form part of an entirely implantable central venous system such as the INFUSAPORT system available from Infusaid Corp., Norwood, MA and the PORT-A-CATH
system available from Pharmacia Laboratories, Piscataway, NJ. These devices are implanted into a subcutaneous pocket under local anesthesia. A catheter, connected to the pump injection port, is threaded through the subclavian vein to the superior vena cava. The implant contains a supply of oligonucleotide in a reservoir which may be replenished as needed by injection of additional drug from a hypodermic needle through a self-sealing diaphragm in the re~ervoir. Completely implantable infusers are preferred, as they are generally well accepted by patients because of the convenience, ease of maintenance and cosmetic advantage of such devices.
:
High dose chemotherapy has been coupled with autologous bone marrow rescue (transplantation) in an attempt to treat melanoma. While high dose chemotherapy has resulted in significantly higher therapeutic responses, patient survival is not generally prolonged:
Jones et al., Cancer Chemother. Pharmacol. 26, 155-6 (1990) (carboplatin, cyclophosphamide and BCNU); Lakhani et al., Br. J. Cancer 61, 330-4 (1990) (melphalan);
Koeppler et al., Onkoloqie 12, 277-9 (1989) (melphalan and BCNU); Thatcher et al., Cancer 63, 1296-302 (1989) (DTIC); Wolff et al., J. Clin. Oncol. 7, 245-9 (1989) (thiotepa); Shea et al., Arch. Dermatol. 124, 878-84 (1988) (cyclophosphamide, cisplatin and carmustine);
Tchekmedian et al., J. Clin. Oncol. 4, 1811-8 (1986) (BCNU, melphalan, or both); Thomas et al., Oncoloav 43, 273-7 (1986) (BCNU and melphalan).
Melanoma, particularly in advanced stages, may be substantially metastatic. Thus, one possible reason for the lack of success of high dose chemotherapy and autologous bone marrow purging in treating melanoma may be a failure to properly purge the harvested marrow of malignant cells which have metastasized to the bone marrow. According to the present invention, c-mYb antisense oligonucleotides may be used as bone marrow purging agents for the in vitro cleansing of bone marrow of melanoma cells in conjunction with high dose conventional chemotherapy. While normal hematopoietic cells are sensitive to c-mYb antisense, they are less sensitive than malignant cells expressing c-~yk, as taught in our copending patent application Serial No.
427,659 and corresponding international application WO90/05445. This differential sensitivity makes possible the use of c-my~ antisense oligonuclQotides in purging bone marrow of neoplastic cells.
According to a method for bone marrow purging, bone marrow is harvested from a donor by standard operating room procedures from t~e iliac bones of the donor.
:
W093/09789 2 1 2 3 fi 1 I PCT/~'S92/0~656 Methods of aspirating bone marrow from donors are well-known in the art. Examples of apparatus and processes for aspirating bone marrow from do~ors are disclosed in U.S. Patents 4,481,946 and 4,486,188, incorporated herein by reference. Sufficient marrow is withdrawn so that the recipient, who is either the donor (autologous transplant) or another individual (allogeneic transplant), may receive from about 4 x 10~ to about 8 x 10d processed marrow cells per kg of bodyweight. This generally requires aspiration of about 750 to about 1000 ml of marrow. The aspirated marrow is filtered until a single cell suspension, known to those skilled in the art as a "buffy coat" preparation, is obtained. This suspension of leukocytes is treated with c-myb antisense lS oligonucleotides in a suitable carrier, advantageously in a concentration of about 8 mg/ml. Alternatively, the leucocyte suspension may be stored in liquid nitrogen using standard procedures known to those skilled in the art until purging is carried out. The purged marrow can be stored frozen in liquid nitrogen until ready for use.
Methods of freezing bone marrow and biological substances are disclosed, for example, in U.S. Patents 4,107,937 and
which is followed by a poly (A) tail of about 14 0 nucleotides.
The antisense oligonucleotides of the invention may be synthesized by any of the known chemical oligonu-cleotide synthesis methods. Such methods are generallydescribed, for example, in Winnacker, From Genes to Clones: Introduction to Gene Technoloqv, VCH Verlagsges-ellschaft mbH (Ibelgaufts trans. 1987). The antisense oligonucleotides are most advantageously prepared by utilizing any of the commercially available, automated nucleic acid synthesizers. One such device, the Applied ~-Biosystems 380B DNA Synthesizer, utilizes ~-cyanoethyl phosphoramidite chemistry.
Since the complete nucleotide synthesis of DNA
complementary to the c-myb mRNA transcript is known, an-tisense oligonucleotides hybridizable with any portion of the mRNA transcript may be prepared by oligonucleotide synthesis methods known to those skilled in the art.
While any length oliqonucleotide may be utilized in the practice of the invention, sequences shorter than 12 bases may be less specific in hybridizing to the target c-mvb mRNA, may be more easily destroyed by enzymatic digestion, and may be destabilized by enzymatic digestion. Hence, oligonucleotides having 12 or more nucleotides are preferred.
Long sequences, particularly sequences longer than about 40 nucleotides, may be somewhat less effective in inhibiting c-myb translation because of decreased uptake by the target cell. Thus, oligomers of 12-40 nucleotides are preferred, more preferably 15-30 nucleotides, most preferably 18-26 nucleotides. While sequences of 18-21 nucleotides are most particularly preferred, for unmodified oligonucleotides, slightly longer chains of up to about 26 nucleotides, are preferred for modified oligonucleot~des such as phosphorothioate oligonucleotides, which hybridize less strongly to mRNA than unmodified oligonucleotides. `
. .
9 ~12~61~
Oligonucleotides complementary to and hybridizable with any portion of the c-myb mRNA
transcript are, in principle, effective for inhibiting translation of the transcript, and capable of inducing the effects herein described. It is believed that translation is most effectively inhibited by blocking the mRNA at a site at or near the initiation codon. Thus, oligonucleotides complementary to the 5'-terminal region of the c-mYb mRNA transcript are preferred. It is believed that secondary or tertiary structure which might interfere with hybridization is minimal in this region.
Moreover, it has been suggested that sequences that are too distant in the 3'-direction from the initiation site may be less effective in hybridizing the mRNA transcripts because of a "read-through" phenomenon whereby the ribosome is postulated to unravel the antisense/sense duplex to permit translation of the message. See, e.g., Shakin, J. Biochemistry 261, 16018 tl986).
The antisense oligonucleotide is preferably directed to a site at or near the initiation codon for protein synthesis. Oligonucleotides complementary to the c-my~ mRNA, including the initiation codon (the first codon at the 5' end of the translated portion of the c-EY~ transcript, comprising nucleotides 114-116 of the complete transcript) are preferred.
While antisense oligomers complementary to the ~'-terminal region of the c-mYb transcript are preferred, particularly the region including the initiation codon, it should be appreciated that usaful antisense oligomers are not limited to those complementary to the sequenceæ
found in the translated portion (nucleotides 114 to 2031) of the mRNA transcript, but also includes oligomers complementary to nucleotide sequences contained in, or exténding into, the 5'-and 3'-untranslated regions.
Oligomers whose complementarity extends into the 5'-untranslated region o~ the c-myb transcript are believed particularly effective in inhibiting c-_y~ translation.
212~611 10 Preferred oligonucleotidescomplementary tot~e 5'-untranslated region of the transcript include molecules having a nucleotide sequence complementary to a portion of the c-mvb mRNA transcript including the cap nucleotide, that is, the nucleotide at the extreme 5'-end of the transcript. The Sl nuclease assay procedure of Molecular Clonina, 2nd edition (Sambrook et al., Eds.
1989), pages 7.66-7.70 (incorporated herein by reference) was essentially followed to map the location of c-~yk cap sites using mRNA isolated from the leukemic cell line CCRF-CEM, which expresses high levels of c-~y~ mRNA. The longest clearly visible band was located 90 base pairs upstream of the published c-mvb cDNA (Majello ç~ al., Proc. ~atl. Acad. Sci. U.S.A., 83, 9536-9640 (1986)), indicating the putative principle cap site. The position of this site is in perfect agreement with the length of the c-myb cDNA cloned from CCRF-CEN cells (Clarke et al., Mol. Cell. Biol., 8, 884-892 (1988)). Sl protection ~; assays also revealed faint bands in addition to the main band corresponding to the cap site. These other bands may represent rare or unstable c-Ey~ mRNA transcripts.
Multiple sites of transcription initiation are not uncommon in genes such as c-myb which lack a perfect TATAA box. The nucleotide sequence of the mRNA
transcript 5'-terminus beginning with the cap nucleotide may be readily established, and antisense oligonuc-leotides complementary and hybridizable thereto may be prepared.
The following 40-mer oligodeoxynucleotide is complementary to the c-mYb mRNA transcript beginning with the initiation codon of the transcript and extending downstream thereof (in the 3' direction): SEQ ID N0:1.
Smaller oligomers based upon the above sequence, in particular, oligomers hybridizable to segments of the c-mvb message containing the initiation codon, may be utilized. Particularly pr~ferrQd are the following 26- to 15-mers: ~
.
W0~3/09789 PCT/US92~09656 1l 2123611 SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ I~ NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SE~ ID N0:12 and SEQ ID NO:13.
Oligodeoxynucleotides complementary to the c-myb mRNA transcript beginning with the second codon of the translated portion of the transcript (nucleotides 117-119 of the complete transcript) are another group of preferred oligomers. Such oligomers include, for example, the following 26- to 15-mers:
SEQ ID N0:14, SE~ ID N0:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23, 3Q SEQ ID N0:24 and SEQ ID N0:25.
The oligonucleotide employed may represent an un-modified or modified oligonucleotide. Thus, oligo-nucleotides hybridizable to the c-~y~ mRNA transcript finding utility according to the present invention include not only oligomers of the biologically sig-W O 93/09789 P ~ tUS92/09656 2 1 ~ 12 -nificant native nucleotides, i.e., A, dA, G, dG, C, dC, T and U, but also oligonucleotide species which have been modified for improved stability and/or lipid solubility~
For example, it i5 known that enhanced lipid solubility and/or resistance to nuclease digestion results by substituting an alkyl group or alkoxy group for a phos-phate oxygen in the internucleotide phosphodiester linkage to form an alkylphosphonate oligonucleoside or alkylphosphotriester oligonucleotide. Non-ionic oligonucleotides such as these are characterized by increased resistance to nuclease hydrolysis and/or increased cellular uptake, while retaining the ability to form stable complexes with complementary nucleic acid sequences. The alkylphosphonates in particular, are stable to nuclease cleavage and soluble in lipid. The preparation of alkylphosphonate oligonucleosides is disclosed in U.S. Patent 4,469,863.
Methylphosphonate oligomers can be prepared by a variety of methods, both in solution and on insoluble polymer supports (Agrawal and Riftina, Nucl. Acids Res., 6, 3009-3024 (1979); Miller et al., Biochemistry, 18, 5134-5142 (1979), Miller et al., J. Biol. Chem., 255, 9659-9665 (1980); Miller et al., Nucl. Acids Res., 11, 5189-5204 (1983), Miller et al., Nucl. Acids Res., 11, 6225-6242 (1983), Miller et al., Biochemistry, 25, 5092-5097 (1986); Engels and Jager, Anqew. Chem. Suppl. 912 (1982); Sinha et al., Tetrahedron Lett. 24, 877-880 (1983); Dorman et al, Tetrahedron, 40, 95~102 tl984);
Jager and Engels, Tetrahedron Lett., 25, 1437-1440 (1984); Noble et al., Nucl. Acids Res., 12, 3387-3404 ~1984); Callahan et al., Proc. Natl. Acad. Sci. USA, 83, 1617-1621 (1986); Koziolkiewicz e~ hemica Scri~ta, 26, 251-260 (1986): Agrawal and Goodchild, Tetrahedron Lett., 38, 3539-3542 (1987); Lesnikowski et ~1-.
Tetrahedron Lett., 28, 5535-5538 (1987); Sarin et al., Proc. Natl. Acad. Sci. USA, 85~ 7448-7451 ~1988)).
The most efficient procedure for preparation of methylphosphonate oligonucleosides involves use of 5'-o-d i m e t h o x y t r i t y l d e o x y n u c l e o s i d e - 3 ' - Q-diisopropylmethylphosphoramidite intermediates, which are similar to the methoxy or ~-cyanoethyl phosphoramidite reagents used to prepare oligodeoxyribonucleotides. The methylphosphonate oligomers can be prepared on controlled pore glass polymer supports using anautomated DNA
synthesizer (Sarin et al., Proc. Natl. Acad. Sci. USA, 85, 7448-7451 (1988)).
Resistance to nuclease digestion may also be achieved by modifying the internucleotide linkage at both the 5' and 3' termini with phosphoroamidites according to the procedure of Dagle et al., Nucl. Acids Res. 18, 4751-4757 (1990).
Phosphorothioate oligonucleotides contain a sulfur-for-oxygen substitution in the internucleotide phosphodiester bond. Phosphorothioate oligonucleotides combine the properties of effective hybridization for duplex formation with substantial nuclease resistance, while retaining the water solubility of a charged phosphate analogue. The charge is believed to confer the property of cellular uptake via a receptor (Loke et al., Proc. Natl. Acad Sci. U.S.A. 86, 3474-3478 (1989)).
Phosphorothioate oligodeoxynucleotide are described by LaPlanche, et al., Nucleic Acids Research 14, 9081 (1986) and by Stec et al., J. Am. Chem. Soc. 106, 6077 (lg84). The general synthetic method for phosphorothio-ate oligonucleotides was modified by Stein et al., Nucl.
Acids Res., 16, 3209-3221 (1988), so that these compounds may readily be synthesized on an automatic synthesizer using the phosphoramidite approach.
Furthermore, recent advances in the production of oligori~onucleotide analogues mean that other agents may also be used for the purposes described here, e.g., 2'-0-methylribonucleotides (Inove et al., ~ucleic Acids Res.
15, 6131 (1987) and chimeric oligonucleotides th,at are W O 93/09789 PC~r/US92/09656 ~ 3 6 1 ~ 14 _ composite RNA-DNA analogues (Inove et al., FEss Lett.
215, 327 (1987).
While inhibition of c-mvb mRNA translation is possible utilizing either antisense oligoribonucleotides or oligodeoxyribonucleotides, free oligoribonucleotides are more susceptible to enzymatic attack by ribonucleases than oligodeoxyribonucleotides. Hence, oligodeoxyribonucleotides are preferred in the practice of the present invention. Oligodeoxyribonucleotides are further preferred because, upon hybridization with c-myb mRNA, the resulting DNA-RNA hybrid duplex is a substrate for RNase H, which specifically attacks the RNA portion of DNA-RNA hybrid. Degradation of the mRNA strand of the duplex releases the antisense oligodeoxynucleotide strand for hybridization with additional c-myb messages.
In general, the antisense oligonucleotides used in the method of the present invention will have a sequence which is completely complementary to the target portion of the c-myb message. Absolute complementarity is not however required, particularly in larger oligomers.
Thus, reference herein to a "nucleotide sequence complementary to at least a portion of the mRNA
transcript" of c-mYb does not necessarily mean a sequence having 100% complementarity with the transcript. In gene-ral, any oligonucleotide having sufficient com-plementarity to form a stable duplex with c-mYb mRNA is suitable. Stable duplex formation depends on the sequence and length of the hybridizing oligonucleotide and the degree of complementarity with the target region of the c-myb message. Generally, the larger the hybridizing oligomer, the more mismatches may be tolerated. More than one mismatch probably will not be tolerated for antisense oligomers of less than about 21 nucleotides. One skilled in the art may readily determine the degree of mismatching which may be tolerated between any given antisense oligomer and the target c-mYb message sequence, based upon the melting wo93/o978s PCT/US92/09656 point, and therefore the stability, of the resulti~g duplex. Melting points of duplexes of a given base pair composition can be readily determined from standard texts, such as Molecular Cloninq: A Laboratory Manual, (2nd edition, 1989), J. Sambrook et al., eds.
While oligonucleotides capable of stable hybridiza-tion with any region of the c-myb message are within the scope of the present invention, oligonucleotides complementary to a region including the initiation codon are believed particularly effective. Particularly preferred are oligonucleotides hybridizable to a region of the c-~y~ mRNA up to 40 nucleotides upstream (in the 5' direction) of the initiation codon or up to 40 nucleotides downstream (in the 3' direction) of that codon.
For therapeutic use, the antisense oligonucleotides may be combined with a pharmaceutical carrier, such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives. The liguid vehicles and excipients are conventional and commercially available.
Illustrative thereof are distilled water, physiological saline, aqueous solution of dextrose, and the like. The c-~yb mRNA antisense oligonucleotides are preferably administeredparenterally,mostpreferably intravenously.
The vehicle is designed accordingly. Alternatively, oligonucleotide may be administered subcutaneously via controlled release dosage forms.
The oligonucleotides may be conjugated to poly(L-lysime) to increase cell penetration. Such conjugates are described by Lemaitre et al., Proc. Natl. Acad. 5ci~
PSA, 84, 648-652 (1987). The procedure requires that the 3'-terminal nucleotide be a ribonucleotide. The resulting aldehyde groups are then randomly coupled to the epsilon-amino groups of lysine residues of poly(L-lysine) by Schiff base formation, and then reduced withsodium cyanoborohydride. This procedure converts the 3'-terminal ribose ring into a morpholine structure antisense oligomers.
In addition to administration with conventional carriers, the antisense oligonucleotides may be administered by a variety of specialized oligonucleotide delivery techniques. For example, oligonucleotides may be encapsulated in for therapeutic delivery. The oligonucleotide, depending upon its solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspension. The hydrophobic layer, generally but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, ionic surfactants such as diacetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature. Oligonucleotides have been successfully encapsulated in unilameller liposomes.
Reconstituted Sendai virus envelopes have been suc-cessfully used to deliver RNA and DNA to cells. Arad et al., Biochem. Biophy. Acta. 859, 88-94 (1986).
The c-mvb antisense oligonucleotides may be administered de novo as the primary therapy.
Alternatively, the oligonucleotides may be administered as an adjuvant following surgical removal of a melanoma to patients who may be disease-free but at high risk of recurrence.
A preferred method of administration of oligonucleotide for treatment of melanoma comprises either regional or systemic perfusion, as is appropriate.
According to a method of regional perfusion, the afferent and efferent vessels supplying the extremity containing the lesion are isolated and connected to a low-flow perfusion pump in continuity with ~n oxygenator and a heat exchanger. The iliac vessels may be used for perfusion of the lower extremity. The axillary vessels are cannulated high in the axilla for upper extremity lesions. Oligonucleotide is added to the perfusion W093/09789 2 1 2 ~ PCT/US92/09656 circuit, and the perfusion is continued for an appropriate time period, e.g., one hour. Perfusion rates of from 100 to 150 ml/minute may be employed for lower extremity lesions, while half that rate should be employed for upper extremity lesions. Systemic heparinization may be used throughout the perfusion, and reversed after the perfusion is complete. This isolation perfusion technique permits administration of higher doses of chemotherapeutic agent than would otherwise be tolerated upon infusion into the arterial or venous sys-temic circulation.
For systemic infusion, the oligonucleotides are preferably delivered via a central venous catheter, which is connected to an appropriate continuous infusion device. Indwelling catheters provide long term access to the intravenous circulation for freguent administration of drugs over extended time periods. They are generally surgically inserted into the external cephalic or internal jugular vein under general or local anesthesia. The subclavian vein is another common site of catheterization. The infuser pump mày be external, or may form part of an entirely implantable central venous system such as the INFUSAPORT system available from Infusaid Corp., Norwood, MA and the PORT-A-CATH
system available from Pharmacia Laboratories, Piscataway, NJ. These devices are implanted into a subcutaneous pocket under local anesthesia. A catheter, connected to the pump injection port, is threaded through the subclavian vein to the superior vena cava. The implant contains a supply of oligonucleotide in a reservoir which may be replenished as needed by injection of additional drug from a hypodermic needle through a self-sealing diaphragm in the re~ervoir. Completely implantable infusers are preferred, as they are generally well accepted by patients because of the convenience, ease of maintenance and cosmetic advantage of such devices.
:
High dose chemotherapy has been coupled with autologous bone marrow rescue (transplantation) in an attempt to treat melanoma. While high dose chemotherapy has resulted in significantly higher therapeutic responses, patient survival is not generally prolonged:
Jones et al., Cancer Chemother. Pharmacol. 26, 155-6 (1990) (carboplatin, cyclophosphamide and BCNU); Lakhani et al., Br. J. Cancer 61, 330-4 (1990) (melphalan);
Koeppler et al., Onkoloqie 12, 277-9 (1989) (melphalan and BCNU); Thatcher et al., Cancer 63, 1296-302 (1989) (DTIC); Wolff et al., J. Clin. Oncol. 7, 245-9 (1989) (thiotepa); Shea et al., Arch. Dermatol. 124, 878-84 (1988) (cyclophosphamide, cisplatin and carmustine);
Tchekmedian et al., J. Clin. Oncol. 4, 1811-8 (1986) (BCNU, melphalan, or both); Thomas et al., Oncoloav 43, 273-7 (1986) (BCNU and melphalan).
Melanoma, particularly in advanced stages, may be substantially metastatic. Thus, one possible reason for the lack of success of high dose chemotherapy and autologous bone marrow purging in treating melanoma may be a failure to properly purge the harvested marrow of malignant cells which have metastasized to the bone marrow. According to the present invention, c-mYb antisense oligonucleotides may be used as bone marrow purging agents for the in vitro cleansing of bone marrow of melanoma cells in conjunction with high dose conventional chemotherapy. While normal hematopoietic cells are sensitive to c-mYb antisense, they are less sensitive than malignant cells expressing c-~yk, as taught in our copending patent application Serial No.
427,659 and corresponding international application WO90/05445. This differential sensitivity makes possible the use of c-my~ antisense oligonuclQotides in purging bone marrow of neoplastic cells.
According to a method for bone marrow purging, bone marrow is harvested from a donor by standard operating room procedures from t~e iliac bones of the donor.
:
W093/09789 2 1 2 3 fi 1 I PCT/~'S92/0~656 Methods of aspirating bone marrow from donors are well-known in the art. Examples of apparatus and processes for aspirating bone marrow from do~ors are disclosed in U.S. Patents 4,481,946 and 4,486,188, incorporated herein by reference. Sufficient marrow is withdrawn so that the recipient, who is either the donor (autologous transplant) or another individual (allogeneic transplant), may receive from about 4 x 10~ to about 8 x 10d processed marrow cells per kg of bodyweight. This generally requires aspiration of about 750 to about 1000 ml of marrow. The aspirated marrow is filtered until a single cell suspension, known to those skilled in the art as a "buffy coat" preparation, is obtained. This suspension of leukocytes is treated with c-myb antisense lS oligonucleotides in a suitable carrier, advantageously in a concentration of about 8 mg/ml. Alternatively, the leucocyte suspension may be stored in liquid nitrogen using standard procedures known to those skilled in the art until purging is carried out. The purged marrow can be stored frozen in liquid nitrogen until ready for use.
Methods of freezing bone marrow and biological substances are disclosed, for example, in U.S. Patents 4,107,937 and
4,117,881.
Other methods of preparing bone marrow for treat-ment with c-mYb antisense may be utilized, which methods may result in even more purified preparations of hemato-poietic cells than the aforesaid buffy coat preparation.
One or more growth factors may be added to the aspirated marrow or buffy coat preparation to stimulate growth of neoplasms, and thereby increase their sensi-tivity to the toxicity of the c-myb antisense oligonucleotides.
After treatment with the antisense oligonucleo-tides, the cells to be transferred are washed with auto-logous plasma or buffer to remove unincorporatedoligomer. The washed cells are then infused back into the patient.
The c-mvb antisense oligonucleotides may b~
administered in a dosage effective for inhibiting the proliferation of melanoma cells in the afflicted individual, while maintaining the viability of normal cells. Such amounts may vary depending on the nature and extent of the neoplasm, the particular oligonucleotide utilized, and other factors. The actual dosage admin-istered may take into account the size and weight of the patient, whether the nature of the treatment is prophy-lactic or therapeutic in nature, the age, health and sexof the patient, the route of administration, whether the treatment is regional or systemic, and other factors.
Inhibition of melanoma cell proliferation has been observed at antisense concentrations of as low as 10 ~g/ml. At 20 ~g/ml, inhibition was profound. Concen-trations of from about 1 to about 100 ~g/ml may be employed, preferably from about 10 ~g/ml to about 100 ~g/ml, most preferably from about 20 ~g/ml to about 60 ~g/ml. The patient should receive a sufficient daily dosage of antisense oligonucleotide to achieve these intercellular concentrations of drug. The daily dosage may range from about 0.1 to 1,000 mg oligonucleotide per day, preferably from about 10 to about 700 mg per day.
Greater or lesser amounts of oliqonucleotide may be administered, as required. Those skilled in the art should be readily able to derive appropriate dosages and schedules of administration to suit the specific circumstance and needs of the patient. Based upon the in vivo study described herein, it is believed that a course of treatment may advantageously comprise infusion of the recommended daily dose of oligonucleotide for a period of from about 3 to about 28 days, more preferably from about 7 to about 10 days. Those skilled in tbe art should readily be able to determine tbe optimal dosage in ach case.
`~.
W093/09789 PCT/US92'09656 For an adult human being, a daily dose of about 3so mg oligonucleotide is believed sufficient, to achieve an effective intercellular concentration of 20 ~g.
For ex vivo antineoplastic application, such as, for example, in bone marrow purging, the c-myb antisense oligonucleotides may be administered in amounts effective to kill neoplastic cells while maintaining the viability of normal hematologic cells. Such amounts may vary depending on the extent to which melanoma cells may have metastasized to the bone marrow, the particular oligonucleotide utilized, the relative sensitivity of t~e neoplastic cells to the oligo-nucleotide, and other factors. Concentrations from about10 to 200 ~g/ml per 105 cells may be employed, preferably from about 40 to 150 ~g/ml per 105 cells. Supplemental dosing of the same or lesser amounts of oligonucleotide are advantageous to optimize the treatment. Thus, for purging bone marrow containinq 2 x 107 cell per ml of marrow volume, dosages of from about 2 to 40 mq antisense per ml of marrow may be effectively utilized, preferably from about 8 to 24 mg/ml. Greater or lesser amounts of oligonucleotide may be employed.
The present invention is described in greater detail in the following non-limiting examples.
Ex~mple 1 Inhibition of CHP Melanoma Growth By c-myb_Antisense Oliaonucleotide CHP melanoma cells (Children's Hospital of Philadelphia, Philadelphia, PA) were grown in RPMI
culture medium containing 2% oxalophosphate and 5% bovine calf serum at 37C in 5% C02. Cells (5,000/ml) were seeded in 200 ~1 volumes into individual wells of a 96 well Costar plate at day -3, and allowed to grow for three days. At this time (day 0), the unmodif~ed antisense 18-mer oligodeoxynucleotide (SEQ ID N0:22) ;~ complementary to codons 2-7 of t~e translated portion of ~, W093~09789 rCT/US92/09656 2 123 6 1~ - 22 -the c-myb mRNA, or the corresponding sense 18-mer (SEQ
ID NO:26), were added to the cultures at concentrations of o, 10, 20, 50 and 100 ~g/ml for 1, 2 or 5 consecutive days. On day 7, 100 ~1 of fresh medium was added to the cultures. Cell viability/proliferation was determined on day 8 utilizing a commercially available kit (CELLTITER 96~ Promega, Madison, WI). The assay is based on the ability of viable cells to convert a tetrazolium salt into a blue formazan product which can be quantified by measuring the absorbance at 570 nm with a conventional microplate reader. The extent of absorbance at 570 nm is proportional to the amount of formazan produced, and thus the number of viable cells remaining. Accordingly, 10 ~1 of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide solution t5 mg/ml) was added to 110 ~1 of cell suspension and incubated for 4 hours at 37-C. 150 ~1 of acidified isopropanol (25 ml of isopropanol plus 0.1 ml of 12 N HCl) was then added and the solution mixed. The optical density was measured at 570 nm. Background from cell debris was eliminated by subtracting a reference measurement at 630 nm.
The results of the viability assay are set forth in Figures 1 (one-day treatment), 2 (two-day treatment) and 3 (five-day treatment). The oligonucleotide concentrations indicated in Figures 2 and 3 are cumulative dosages for the two- and five-day treatments, respectively. It may be appreciated from the figures that treatment of the melanoma cells with antisense oligomer resulted in sequence-specific killing. The effect was most pronounced when the melanoma cells were treated for one day with an antisense oligomer concen-tration of at least 50 ~g/ml, or when the cells were treated on consecutive days with as little as 20 ~g/ml oligomer.
W O 93/09789PC~r/U~92/09656 ExamPle 2 Inhibition of SK MEL-37 Melanoma By c-mvb Antisense Oliqonucleotide 5The effect of c-mYb antisense oligonucleotide on another human melanoma line, SK MEL-37 (Sloan Kettering Institute, New York, NY) was determined. Cells were treated according to the procedure of Example 1 on day 0 with the same sense and antisense oligomers, in the 10 same concentrations. Cell viability was assayed on day
Other methods of preparing bone marrow for treat-ment with c-mYb antisense may be utilized, which methods may result in even more purified preparations of hemato-poietic cells than the aforesaid buffy coat preparation.
One or more growth factors may be added to the aspirated marrow or buffy coat preparation to stimulate growth of neoplasms, and thereby increase their sensi-tivity to the toxicity of the c-myb antisense oligonucleotides.
After treatment with the antisense oligonucleo-tides, the cells to be transferred are washed with auto-logous plasma or buffer to remove unincorporatedoligomer. The washed cells are then infused back into the patient.
The c-mvb antisense oligonucleotides may b~
administered in a dosage effective for inhibiting the proliferation of melanoma cells in the afflicted individual, while maintaining the viability of normal cells. Such amounts may vary depending on the nature and extent of the neoplasm, the particular oligonucleotide utilized, and other factors. The actual dosage admin-istered may take into account the size and weight of the patient, whether the nature of the treatment is prophy-lactic or therapeutic in nature, the age, health and sexof the patient, the route of administration, whether the treatment is regional or systemic, and other factors.
Inhibition of melanoma cell proliferation has been observed at antisense concentrations of as low as 10 ~g/ml. At 20 ~g/ml, inhibition was profound. Concen-trations of from about 1 to about 100 ~g/ml may be employed, preferably from about 10 ~g/ml to about 100 ~g/ml, most preferably from about 20 ~g/ml to about 60 ~g/ml. The patient should receive a sufficient daily dosage of antisense oligonucleotide to achieve these intercellular concentrations of drug. The daily dosage may range from about 0.1 to 1,000 mg oligonucleotide per day, preferably from about 10 to about 700 mg per day.
Greater or lesser amounts of oliqonucleotide may be administered, as required. Those skilled in the art should be readily able to derive appropriate dosages and schedules of administration to suit the specific circumstance and needs of the patient. Based upon the in vivo study described herein, it is believed that a course of treatment may advantageously comprise infusion of the recommended daily dose of oligonucleotide for a period of from about 3 to about 28 days, more preferably from about 7 to about 10 days. Those skilled in tbe art should readily be able to determine tbe optimal dosage in ach case.
`~.
W093/09789 PCT/US92'09656 For an adult human being, a daily dose of about 3so mg oligonucleotide is believed sufficient, to achieve an effective intercellular concentration of 20 ~g.
For ex vivo antineoplastic application, such as, for example, in bone marrow purging, the c-myb antisense oligonucleotides may be administered in amounts effective to kill neoplastic cells while maintaining the viability of normal hematologic cells. Such amounts may vary depending on the extent to which melanoma cells may have metastasized to the bone marrow, the particular oligonucleotide utilized, the relative sensitivity of t~e neoplastic cells to the oligo-nucleotide, and other factors. Concentrations from about10 to 200 ~g/ml per 105 cells may be employed, preferably from about 40 to 150 ~g/ml per 105 cells. Supplemental dosing of the same or lesser amounts of oligonucleotide are advantageous to optimize the treatment. Thus, for purging bone marrow containinq 2 x 107 cell per ml of marrow volume, dosages of from about 2 to 40 mq antisense per ml of marrow may be effectively utilized, preferably from about 8 to 24 mg/ml. Greater or lesser amounts of oligonucleotide may be employed.
The present invention is described in greater detail in the following non-limiting examples.
Ex~mple 1 Inhibition of CHP Melanoma Growth By c-myb_Antisense Oliaonucleotide CHP melanoma cells (Children's Hospital of Philadelphia, Philadelphia, PA) were grown in RPMI
culture medium containing 2% oxalophosphate and 5% bovine calf serum at 37C in 5% C02. Cells (5,000/ml) were seeded in 200 ~1 volumes into individual wells of a 96 well Costar plate at day -3, and allowed to grow for three days. At this time (day 0), the unmodif~ed antisense 18-mer oligodeoxynucleotide (SEQ ID N0:22) ;~ complementary to codons 2-7 of t~e translated portion of ~, W093~09789 rCT/US92/09656 2 123 6 1~ - 22 -the c-myb mRNA, or the corresponding sense 18-mer (SEQ
ID NO:26), were added to the cultures at concentrations of o, 10, 20, 50 and 100 ~g/ml for 1, 2 or 5 consecutive days. On day 7, 100 ~1 of fresh medium was added to the cultures. Cell viability/proliferation was determined on day 8 utilizing a commercially available kit (CELLTITER 96~ Promega, Madison, WI). The assay is based on the ability of viable cells to convert a tetrazolium salt into a blue formazan product which can be quantified by measuring the absorbance at 570 nm with a conventional microplate reader. The extent of absorbance at 570 nm is proportional to the amount of formazan produced, and thus the number of viable cells remaining. Accordingly, 10 ~1 of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide solution t5 mg/ml) was added to 110 ~1 of cell suspension and incubated for 4 hours at 37-C. 150 ~1 of acidified isopropanol (25 ml of isopropanol plus 0.1 ml of 12 N HCl) was then added and the solution mixed. The optical density was measured at 570 nm. Background from cell debris was eliminated by subtracting a reference measurement at 630 nm.
The results of the viability assay are set forth in Figures 1 (one-day treatment), 2 (two-day treatment) and 3 (five-day treatment). The oligonucleotide concentrations indicated in Figures 2 and 3 are cumulative dosages for the two- and five-day treatments, respectively. It may be appreciated from the figures that treatment of the melanoma cells with antisense oligomer resulted in sequence-specific killing. The effect was most pronounced when the melanoma cells were treated for one day with an antisense oligomer concen-tration of at least 50 ~g/ml, or when the cells were treated on consecutive days with as little as 20 ~g/ml oligomer.
W O 93/09789PC~r/U~92/09656 ExamPle 2 Inhibition of SK MEL-37 Melanoma By c-mvb Antisense Oliqonucleotide 5The effect of c-mYb antisense oligonucleotide on another human melanoma line, SK MEL-37 (Sloan Kettering Institute, New York, NY) was determined. Cells were treated according to the procedure of Example 1 on day 0 with the same sense and antisense oligomers, in the 10 same concentrations. Cell viability was assayed on day
5. The results are shown in Figure 4.
Again, treatment of melanoma cells with a single 50 ~g/ml dose of antisense oligomer was sufficient to induce substantial cell killing, in comparison to sense-15 treated or untreated cells.
Example 3 Inhibition of SK MEL-37 Melanoma Growth in vivo By c-myb Antisense Oligonucleotide The effect of c-myb antisense oligonucleotide on the growth of melanoma cells n vivo was investigated.
250,000 melanoma cells (SK NEL-37) were injected subcutaneously into each of four severe combined 25 immunodeficient mice (C.B-17/LCRTAC-SCID DF from Fox Chase Cancar Institute, Philadelphia PA) at day -21. The tumors were allowed to grow to a size of approximately 5 mm in diameter. On day 1, ALZET constant infusion pumps ~Alza Corporation, Palo Alto, CA) delivering a 30 total dosage of 700 ~g oli~omer over 7 days were surgically implanted into the mice. The tumor area was then monitored daily. One mouse received no oligomer.
Another mouse received a 24-mer "sense" phosphorothioate oligonucleotide (corresponding to codons 2-~ of the 35 translated c-~yb mRNA). Two mice received a 24-mer anti-sense phosphorothioate oligonucleotide having the nucleotide sequence of SEQ ID NO: 16 (TATGCTGTGC CGGGGGT- r CTTC GGGC), complementary to c-~yk mRNA codons 2-9. The results are shown in Figure 5. Each curve represents one 212361~ - 24 -animal. In the two antisense-treated animals, the tumor size stayed the same or regressed. In contrast, the tumors continued to grow in the sense-treated and control animals.
The animals were sacrificed on day 19. The tumors were removed and weighed. The weights of the tumors in the control and sense-treated animals were 9.9 and 9.6 grams, respectively. The tumor weights in the two anti-sense treated animals were only 0.1 and 0.2 grams.
The following non-limiting example illustrates one methodology for purging bone marrow of metastatic melanoma.
Exampl~ ~
Bone Marrow Purqinq with c-myb Antisense Oliqonucleotide Bone marrow is harvested from the iliac bones of a donor under general anesthesia in an operating room using standard techniques. Multiple aspirations are taken into heparinized syringes. Suf f icient marrow is withdrawn so that the marrow recipient will be able to receive about 4 x 108 to about 8 x 10~ processed marrow cells per kg of body weight. Thus, about 750 to 1000 ml of marrow is withdrawn. The aspirated marrow is transferred immediately into a transport medium (TC-l99, Gibco, Grand Island, New York) containing 10,000 units of preservative-free heparin per 100 ml of medium. The aspirated marrow is filtered through three progressively finer meshes until a single cell suspension results, i.e., a suspension devoid of cellular aggregates, debris and bone particles. The filtered marrow is then processed further into an automated cell separator (e.g., Cobe 2991 Cell Processor) which prepares a "buffy coat"
product, (i.e., leukocytes devoid of red cells and platelets). The buffy coat preparation is then placed in a transfer pack for further processing and storage.
It may be stored until purging in liquid nitrogen using standard procedures. Alternatively, purging can be - 25 21 2~ 61 1 carried out immediately, then the purged marrow may be stored frozen in liquid nitrogen until it is ready for transplantation.
The purging procedure may be carried out as follows. Cells in the buffy coat preparation are adjusted to a cell concentration of about 2 x 10~/ml in TC-199 containing about 20% autologous plasma. C-~y~
antisense oligodeoxynucleotide, for example, in a concentration of about 8 mg/ml, is added to the transfer packs containing the cell suspension. Recombinant human hematopoietic growth factors, e.g., rH IL-3 or rH GM-CSF, may be added to the suspension to stimulate growth of neoplasms and thereby increase their sensitivity c-mvb antisense oligonucleotide toxicity. The transfer packs are then placed in a 37-C waterbath and incubated for 18 - 24 hours with gentle shaking. The cells may then either be frozen in liquid nitrogen or washed once at 4-C
in TC-l99 containing about 20% autologous plasma to remove unincorporated oligomer. Washed cells are then infused into the recipient. Care must be taken to work under sterile conditions wherever possible and to ; maintain scrupulous aseptic techniques at all times.
The present invention may be embodied in other spe-cific forms without departing from the spirit or essen-tial attributes thereof and, accordingly, referenceshould be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.
All references cited herein with respect to synthetic, preparative and analytical procedures are incorporated by reference.
W093/09789 PCTiUS92/096~6 SEOUENCE LISTING
(1) GENERAL INFORNATION: ..
(i) APPLICANT: TEMPLE UNIVERSITY - OF THE
S COMMONWEALTH SYSTEM OF HIGHER EDUCATION
(a~ INVENTOR8: Gewirtz, Alan M.
Calabretta, Bruno (ii) TITLE OF INVENTION: Treatment of Melanoma with Antisense Oligonucleotides to c-~y~ Proto-oncogene.
(iii) NUMBER OF 8EQUENCE8: 26 (iv) CORR~8PONDENCE ADDRE88:
(A) ADDR~88B~: Temple University - of the Common-wealth System of Higher Education (B) 8TRBET: 406 University Services Building ~:
(C) CITY: Philadelphia (D) BTATE: Pennsylvania :.
(E) QO~NTRY: U.S.A.
(F) ~IP: 19122 (v) COMPUTER READABLE FORH: `-(A) MEDI~M TYPE: Diskette, 3.50 inch, 720 Kb (B) COMP~TER: IBM PS/2 (C) OP~RATING 8Y8TEM: MS-DOS
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(B) FILING DATE:
(C) CLAS8IFICATION:
(vii) PRIORITY APP~ICATION DATA:
(A) APPLICATION NUMBER: U.S. Application Serial No. 792,999 (B) FILING DAT~: 15 November 1991 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAMES Monaco, Daniel A.
~B) R~I8~RATION N~NBER: 30,480 (C) ~FER~NCg/DOC~ET N~MBERs 60S6-159 PCT 1 (ix) T~COMNnNICATION INFORNATION:
; (A) TE~EP~ONE: (215) 568-8383 W O 93/09789 PC~r/US92/~9656 - 27 _ 2 1 2 ~ 6 1 1 :
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(A) LENGTH: 17 Nucleotides (B) TYPE: nucleic acid (C) 8TRANDEDNE88: single stranded (D) TOPO~OGY: linear (xi) 8EQ~ENCE DE8CRIPTION: 8EQ ID NO:23:
(2) INFORMATION FOR SEQ ID NO:24:
(i) 8EQ~ENCE CH~RACTERISTIC8:
(A) LENGTH: 16 Nucleotides ~B) TYPE: nucleic acid tc3 8TRANDEDNE88: single stranded (D) TOPO~OGY: linear (xi) 8BQUENCE DE8CRIPTION: 8EQ ID NO:24:
(2) INFORMATION FOR 8EQ ID NO:25:
(i) 8EQ~ENC~ CXARACTERI8TIC8s (A) LENGTH: 15 Nucleotides (B) TYPE: nucleic acid W O 93/09789 2 1 2 3 6 1 1 PC~r/US92/09656 (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (xi) 8EQUENCE DESCRIPTION: SEQ ID NO:25:
(2) INFORNATION FOR 8EQ ID NO:26:
(i) 8EQUENCE CHARACTERI8TICS:
(A) LENGTH: 18 Nucleotides (B) TYPE: nucleic acid (C) 8TRANDEDNESS: single stranded (D) TOPOLOGY: linear (xi) SEQUENCB DESCRIPTION: 8EQ ID NO:26:
Again, treatment of melanoma cells with a single 50 ~g/ml dose of antisense oligomer was sufficient to induce substantial cell killing, in comparison to sense-15 treated or untreated cells.
Example 3 Inhibition of SK MEL-37 Melanoma Growth in vivo By c-myb Antisense Oligonucleotide The effect of c-myb antisense oligonucleotide on the growth of melanoma cells n vivo was investigated.
250,000 melanoma cells (SK NEL-37) were injected subcutaneously into each of four severe combined 25 immunodeficient mice (C.B-17/LCRTAC-SCID DF from Fox Chase Cancar Institute, Philadelphia PA) at day -21. The tumors were allowed to grow to a size of approximately 5 mm in diameter. On day 1, ALZET constant infusion pumps ~Alza Corporation, Palo Alto, CA) delivering a 30 total dosage of 700 ~g oli~omer over 7 days were surgically implanted into the mice. The tumor area was then monitored daily. One mouse received no oligomer.
Another mouse received a 24-mer "sense" phosphorothioate oligonucleotide (corresponding to codons 2-~ of the 35 translated c-~yb mRNA). Two mice received a 24-mer anti-sense phosphorothioate oligonucleotide having the nucleotide sequence of SEQ ID NO: 16 (TATGCTGTGC CGGGGGT- r CTTC GGGC), complementary to c-~yk mRNA codons 2-9. The results are shown in Figure 5. Each curve represents one 212361~ - 24 -animal. In the two antisense-treated animals, the tumor size stayed the same or regressed. In contrast, the tumors continued to grow in the sense-treated and control animals.
The animals were sacrificed on day 19. The tumors were removed and weighed. The weights of the tumors in the control and sense-treated animals were 9.9 and 9.6 grams, respectively. The tumor weights in the two anti-sense treated animals were only 0.1 and 0.2 grams.
The following non-limiting example illustrates one methodology for purging bone marrow of metastatic melanoma.
Exampl~ ~
Bone Marrow Purqinq with c-myb Antisense Oliqonucleotide Bone marrow is harvested from the iliac bones of a donor under general anesthesia in an operating room using standard techniques. Multiple aspirations are taken into heparinized syringes. Suf f icient marrow is withdrawn so that the marrow recipient will be able to receive about 4 x 108 to about 8 x 10~ processed marrow cells per kg of body weight. Thus, about 750 to 1000 ml of marrow is withdrawn. The aspirated marrow is transferred immediately into a transport medium (TC-l99, Gibco, Grand Island, New York) containing 10,000 units of preservative-free heparin per 100 ml of medium. The aspirated marrow is filtered through three progressively finer meshes until a single cell suspension results, i.e., a suspension devoid of cellular aggregates, debris and bone particles. The filtered marrow is then processed further into an automated cell separator (e.g., Cobe 2991 Cell Processor) which prepares a "buffy coat"
product, (i.e., leukocytes devoid of red cells and platelets). The buffy coat preparation is then placed in a transfer pack for further processing and storage.
It may be stored until purging in liquid nitrogen using standard procedures. Alternatively, purging can be - 25 21 2~ 61 1 carried out immediately, then the purged marrow may be stored frozen in liquid nitrogen until it is ready for transplantation.
The purging procedure may be carried out as follows. Cells in the buffy coat preparation are adjusted to a cell concentration of about 2 x 10~/ml in TC-199 containing about 20% autologous plasma. C-~y~
antisense oligodeoxynucleotide, for example, in a concentration of about 8 mg/ml, is added to the transfer packs containing the cell suspension. Recombinant human hematopoietic growth factors, e.g., rH IL-3 or rH GM-CSF, may be added to the suspension to stimulate growth of neoplasms and thereby increase their sensitivity c-mvb antisense oligonucleotide toxicity. The transfer packs are then placed in a 37-C waterbath and incubated for 18 - 24 hours with gentle shaking. The cells may then either be frozen in liquid nitrogen or washed once at 4-C
in TC-l99 containing about 20% autologous plasma to remove unincorporated oligomer. Washed cells are then infused into the recipient. Care must be taken to work under sterile conditions wherever possible and to ; maintain scrupulous aseptic techniques at all times.
The present invention may be embodied in other spe-cific forms without departing from the spirit or essen-tial attributes thereof and, accordingly, referenceshould be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.
All references cited herein with respect to synthetic, preparative and analytical procedures are incorporated by reference.
W093/09789 PCTiUS92/096~6 SEOUENCE LISTING
(1) GENERAL INFORNATION: ..
(i) APPLICANT: TEMPLE UNIVERSITY - OF THE
S COMMONWEALTH SYSTEM OF HIGHER EDUCATION
(a~ INVENTOR8: Gewirtz, Alan M.
Calabretta, Bruno (ii) TITLE OF INVENTION: Treatment of Melanoma with Antisense Oligonucleotides to c-~y~ Proto-oncogene.
(iii) NUMBER OF 8EQUENCE8: 26 (iv) CORR~8PONDENCE ADDRE88:
(A) ADDR~88B~: Temple University - of the Common-wealth System of Higher Education (B) 8TRBET: 406 University Services Building ~:
(C) CITY: Philadelphia (D) BTATE: Pennsylvania :.
(E) QO~NTRY: U.S.A.
(F) ~IP: 19122 (v) COMPUTER READABLE FORH: `-(A) MEDI~M TYPE: Diskette, 3.50 inch, 720 Kb (B) COMP~TER: IBM PS/2 (C) OP~RATING 8Y8TEM: MS-DOS
(D) 80FTWARE: WordPerfect 5.1 tvi) CURRENT APPLICATION DATA:
: 25 (A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLAS8IFICATION:
(vii) PRIORITY APP~ICATION DATA:
(A) APPLICATION NUMBER: U.S. Application Serial No. 792,999 (B) FILING DAT~: 15 November 1991 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAMES Monaco, Daniel A.
~B) R~I8~RATION N~NBER: 30,480 (C) ~FER~NCg/DOC~ET N~MBERs 60S6-159 PCT 1 (ix) T~COMNnNICATION INFORNATION:
; (A) TE~EP~ONE: (215) 568-8383 W O 93/09789 PC~r/US92/~9656 - 27 _ 2 1 2 ~ 6 1 1 :
(B) TELEFAX: (215) 568-5549 (C) TELEX: None (2) INFORMATION FOR 8EQ ID NO:l:
(i) SEQUENCB CHARACTERISTICS:
(A) LENG~H: 40 Nucleotides (B) TYPE: nucleic acid (C) BTRANDEDNESS: single stranded (D) TOPOLOGY: linear (xi) 8EQ~ENCE DESCRIPTION: 8EQ ID NO:l:
(2) INFO~MATION FOR 8EQ ID NO:2:
(i) 8EQUENCE C~ARACTERI8TIC8:
(A) BENGTH: 26 Nucleotides tB) TYPB: nucleic acid (C) 8TRANDEDNE88: single stranded :~
(D) TOPOLOGY: linear (xi) 8EQ~ENC~ DE8CRIPTION: 8EQ ID NO:2:
(2) INFORNATION FOR 8EQ ID NO:3:
(i) 8EQUENCE CHARACTERISTIC8:
(A) ~ENGTH: 25 Nucleotides (B) TYPB: nucleic acid (C) 8TRANDEDNE88: single stranded (D) TOPOLOGY: linear (xi) 8EQUENC~ DESCRIPTION: SEQ ID NO:3:
(2) INFORMATION FOR 8EQ ID NO:4:
(i) 8BQUENCB CHARACTERI8TIC8~
(A) ~ENGT~: 24 Nucleotides (B) TYP~s nucleic acid (C) 8TRAND~DNE88: single stranded (D) ~OPO~OGYs linear (Xi) 8EQ~NCE DE8CRIPTION: 8EQ ID NO:4:
Wo93/n97x9 PCT/US92/09656 2 123 6 li 28 -(2) INFORMATION FOR SEQ ID NO:5:
(i) 8EQUENCE CHARACTERISTICS:
(A) LENGTH: 23 Nucleotides (B) TYPE: nucleic acid (C) 8TRANDEDNEg8: single stranded (D) TOPOLOGY: linear (xi) 8EQUENCE DESCRIPTION: SEQ ID NO:5:
- CTGTGCCGGG GTCTTCGGGC CAT 23 :
(2) INFO~NATION FOR 8EQ ID NO:6:
(i) 8EQUENCE CHARACTERISTIC8:
(A) LENGTH: 22 Nucleotides (B) TYPE: nucleic acid (C) 8TRANDEDNE88: single stranded (D) TOPOLOGY: linear (xi) 8EQUENCE DESCRIPTION: 8EQ ID NO:6:
(2) INFORMATION FOR 8EQ ID NO:7:
(i) 8EQUENCB CHARACTERI8TIC8:
(A) LENGTH: 21 Nucleotides (B) TYPE: nucleic acid (C) 8TRANDEDNE8B: single stranded (D) TOPOLOGY: linear (xi) 8EQUENCE DESCRIPTION: SEQ ID NO:7:
(2) INFORMATION FOR 8EQ ID NO:8:
(i) 8EQ~ENCB CHARACTERI8TICS:
(A) L~NGTH: 20 Nucleotides (B) TYPB: nucleic acid (C) 8TRANDEDNE88s single stranded (~) TOPO~O~Y: linear (xi) 8EQ~BNC~ DB8CaIPTION: BBQ ID NO~8:
3~ TGCCGGGGTC TTCGGGCCAT 20 - 29 2I ~ ~ 6 (2) INFORMATION FOR SEQ ID NO:9:
(i) 8EQUENCE CHARACTERISTICS:
(A) LENGT~: 19 Nucleotides (B) TYPE: nucleic acid (C) 8TRANDEDNE88: sinqle stranded (D) TOPOLOGY: linear (xi) 8BQUBNCB DE8CRIPTION: 8EQ ID NO:9:
(2) INFORMATION FOR 8EQ ID NO:10:
(i) 8EQUBNCE CaARACTERI8TIC8:
(A) LENGTH: 18 Nucleotides (B) TYPB: nucleic acid (C) 8TRANDBDNE88: single stranded (D) TOPOLOGY: linear (xi) 8BQ~BNCB DESCRIPTION: 8EQ ID NO:lO:
. (2) INFO~MATION FOR 8BQ ID NO:ll:
(i) 8BQ~NCE CHARACTERI8TIC8:
:: (A) LENGT~: 17 Nucleotides (B) TYPE: nucleic acid (C) 8TRUNDEDNE88: single stranded (D) TOPOLOGY: linear (xi) 8EQUENCE DEæCRIPTION: 8EQ ID NO:ll:
(2) INFORNATION FOR 8EQ ID NO:12:
(i) 8EQ~ENC~ CHARACTERI8TICS:
(A) LENGTH: 16 Nucleotides (B) TYPEs nucleic acid tC) 8~RUNDEDNE88: sinqle strand~d (D~ ~oPo~oays linear (Xi) 8~Q~NCE D~8CRIPTIONs 8EQ ID NOs12:
2) INFORNATION FOR 8~Q ID NO:13:
.~:
W093/097Xg PCT/US92/09656 212~.611 30 ~
(i) 8EQUENCE CHARACTERISTICS:
(A) LENGTH: 15 Nucleotides (B) TYP~: nucleic acid (C) 8TRANDEDNES8: single stranded (D) TOPOLOGY: linear (xi) 8EQUENCE DE8CRIPTION: SEQ ID NO:13:
; (2) INFORMATION FOR 8BQ ID NO:14:
(i) 8EQU~NCE CHARACTERI8TIC8:
(A) ~ENGTH: 26 Nucleotides (B) TYP~: nucleic acid ; (C) 8TRANDEDNE88: single stranded (D) TOPOLOGY: linear (xi) 8EQ~ENCE DE8CRIPTION: 8EQ ID NO:14:
(2) INFORMATION FOR 8EQ ID NO:15:
~:: (i) 8EQUENCE CHARACTBRI8TIC8:
(A) LENGTH: 25 Nucleotides (B) TYeB nucleic acid :
:~: (C) 8ThANDEDNE88: single stranded : (D) TO~OLOGY: linear (xi) 8BQUENCE DESCRIPTION: 8EQ ID NO:15:
~2) INFORM~TION FOR 8EQ ID NO:16:
(i) 8EQUENCB CHaRACTERI8TIC8:
(A) L~NGT~: 24 Nucleotides (B) TYPE: nucleic acid (C) 8TRANDEDNE88s singlQ stranded (D) TOPOLOGYs linear (Xi) 8~Q~NCE DE8C~IP~ION: 8EQ lD NOs16:
: TATGCTGTGC CGGGGTCTTC GGGC 24 : 35 ~ (2) INFOaMATION FOR 8EQ ID NO:17:
~, :
~ (i) 8EQ~ENCE CHARACTBRI8TIC8:
W O 93/097X9 PC~r/US92/096~6 3, 2 1 2~ 61 1 (A) LENGTH: 23 Nucleotides (B) TYPE: nucleic acid (C) 8TRANDEDNESS: single stranded (D) TOPOLOGY: linear (xi) 8EQVENCE DESCRIPTION: SEQ ID NO:17:
(2) INFORNATION FO% 8EQ ID NO:18:
(i) ~EQVENCE CHARACTERI8TICS:
(A) LENGTH: 22 Nucleotides (B) TYPE: nucleic acid (C) 8TRANDEDNE88: single stranded (D) TOPO~OGY: linear (xi) 8EQV~NCE DE8CRIPTION: ~EQ ID NO:18:
(2) INFORMATION FOR 8EQ ID NO:19:
(i) 8EQVENCE CHARACTERI8TIC8:
(A) L~NGTH: 21 Nucleotides ~B) TYPB: nucleic acid (C) 8TRANDEDNES8: single stranded (D) TOPOLOGY: linear txi) 8EQUENCE DESCRIPTION: SEQ ID NO:l9:
(2) INFORNATION FOR SEQ ID NO:20:
(i) 8EQ~ENCE CHARACTERI8TIC8:
(A) LENGT~: 20 Nucleotides (B) TYPE: nucleic acid (C) BT~ANDEDNE88: single stranded (D) TOPO~OGY: linear (xi) 8EQVENCE DE8CRIPTION: 8EQ ID NO:20:
(2) INFORMATION FOR 8EQ ID NO:21:
(i) 8~Q~ENC~ C~ARACTERI8TIC8:
~: tA) LENGTH: 19 Nucleotides '~ 1 2 ~ 32 -(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (xi) 8EQUENCE DESCRIPTION: SEQ ID NO:21:
(2) INFORNATION FOR ~EQ ID NO:22:
(i) 8EQUENC~ CHARACTERI8TIC8:
(A) LENGTH: 18 Nucleotides (B) TYPE: nucleic acid (C) 8TRANDEDNE8B: single stranded (D) TOPOLOGY: linear (xi) 8EQUENCE DE8CRIPTION: 8EQ ID NO:22:
(2) INFORMATION FOR SEQ ID NO:23:
(i) 8EQUENCE CXARACTERISTICS:
(A) LENGTH: 17 Nucleotides (B) TYPE: nucleic acid (C) 8TRANDEDNE88: single stranded (D) TOPO~OGY: linear (xi) 8EQ~ENCE DE8CRIPTION: 8EQ ID NO:23:
(2) INFORMATION FOR SEQ ID NO:24:
(i) 8EQ~ENCE CH~RACTERISTIC8:
(A) LENGTH: 16 Nucleotides ~B) TYPE: nucleic acid tc3 8TRANDEDNE88: single stranded (D) TOPO~OGY: linear (xi) 8BQUENCE DE8CRIPTION: 8EQ ID NO:24:
(2) INFORMATION FOR 8EQ ID NO:25:
(i) 8EQ~ENC~ CXARACTERI8TIC8s (A) LENGTH: 15 Nucleotides (B) TYPE: nucleic acid W O 93/09789 2 1 2 3 6 1 1 PC~r/US92/09656 (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (xi) 8EQUENCE DESCRIPTION: SEQ ID NO:25:
(2) INFORNATION FOR 8EQ ID NO:26:
(i) 8EQUENCE CHARACTERI8TICS:
(A) LENGTH: 18 Nucleotides (B) TYPE: nucleic acid (C) 8TRANDEDNESS: single stranded (D) TOPOLOGY: linear (xi) SEQUENCB DESCRIPTION: 8EQ ID NO:26:
Claims (59)
1. A method for the treatment of melanoma com-prising administering to an individual in need of such treatment an effective amount of an oligonucleotide which has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-myb gene, said oligonucleotide being hybridizable to said mRNA
transcript.
transcript.
2. A method according to claim 1 wherein the oligonucleotide is an at least 12-mer.
3. A method according to claim 2 wherein the oligonucleotide is a methylphosphonate oligonucleoside or phosphorothioate oligonucleotide.
4. A method according to claim 2 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the c-myb mRNA lying within about 40 nucleotides of the translation initiation codon.
5. A method according to claim 2 wherein the oligonucleotide is an oligodeoxynucleotide having a deoxynucleotide sequence complementary to a portion of the c-myb mRNA transcript including the translation initiation codon of said transcript and/or the codon immediately downstream from the initiation codon.
6. A method according to claim 2 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oligodeoxynucleotide.
7. A method according to claim 6 wherein the oligonucleotide is a methylphosphonate oligonucleoside or a phosphorothioate oligonucleotide.
8. A method according to claim 6 wherein the oligonucleotide is from a 15-mer to 30-mer.
9. A method according to claim 8 wherein the oligonucleotide is from a 18-mer to 26-mer.
10. A method according to claim 9 wherein the oligonucleotide is from a 18-mer to 21-mer.
11. A method according to claim 8 wherein the oligonucleotide is an oligodeoxynucleotide selected from the group consisting of:
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10.
SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10.
SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.
12. A method according to claim 8 wherein the oligonucleotide is an oligodeoxynucleotide selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.
13. A method according to claim 12 wherein the oligonucleotide comprises SEQ ID NO:22.
14. A method according to claim 8 wherein the oligonucleotide is a phosphorothioate oligodeoxynucleotide or methylphosphonate oligodeoxynucleoside having a nucleotide/nucleoside sequence corresponding to any of the following:
SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15 SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 or SEQ ID NO:25.
SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15 SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 or SEQ ID NO:25.
15. A method according to claim 14 wherein the oligonucleotide comprises a phosphorothioate oligodeoxynucleotide.
16. A method according to claim 15 wherein the phosphorothioate oligodeoxynucleotide has a nucleotide sequence corresponding to SEQ ID NO:16.
17. A method for purging bone marrow of metastasized melanoma cells comprising treating bone marrow cells aspirated from a melanoma-inflicted individual with an effective amount of an oligonucleotide which has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-myb gene, said oligonucleotide being hybridizable to said mRNA transcript, and returning the thus-treated cells to the body of the afflicted individual.
18. A method according to claim 17 wherein the oligonucleotide is an at least 12-mer.
19. A method according to claim 18 wherein the oligonucleotide is a methylphosphonate oligonucleoside or phosphorothioate oligonucleotide.
20. A method according to claim 18 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the c-myb mRNA lying within about 40 nucleotides of the translation initiation codon.
21. A method according to claim 18 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oligodeoxynucleotide.
22. A method according to claim 21 wherein the oligonucleotide is from a 15-mer to 30-mer.
23. A method according to claim 22 wherein the oligonucleotide is from a 18-mer to 26-mer.
24. A method according to claim 23 wherein the oligonucleotide is from a 18-mer to 21-mer.
25. A method according to claim 22 wherein the oligonucleotide is an unmodified oligodeoxynucleotide, phosphorothioate oligodeoxynucleotide or methylphosphonate oligodeoxynucleoside having a nucleotide/nucleoside sequence selected from the group consisting of:
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.
26. A method according to claim 25 wherein the oligonucleotide has the nucleotide sequence SEQ ID NO:22.
27. A method according to claim 25 wherein the oligonucleotide has the nucleotide sequence SEQ ID NO:16.
28. Use of an oligonucleotide which has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-myb gene, said oligonucleotide being hybridizable to said mRNA
transcript, for the manufacture of a medicament for treatment of melanoma.
transcript, for the manufacture of a medicament for treatment of melanoma.
29. Use according to claim 28 wherein the oligonucleotide is an at least 12-mer.
30. Use according to claim 29 wherein the oligonucleotide is a methylphosphonate oligonucleoside or phosphorothioate oligonucleotide.
31. Use according to claim 29 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the c-myb mRNA lying within about 40 nucleotides of the translation initiation codon.
32. Use according to claim 29 wherein the oligonucleotide is an oligodeoxynucleotide having a deoxynucleotide sequence complementary to a portion of the c-myb mRNA transcript including the translation initiation codon of said transcript and/or the codon immediately downstream from the initiation codon.
33. Use according to claim 29 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oligodeoxynucleotide.
34. Use according to claim 33 wherein the oligonucleotide is a methylphosphonate oligonucleoside or a phosphorothioate oligonucleotide.
35. Use according to claim 33 wherein the oligonucleotide is from a 15-mer to 30-mer.
36. Use according to claim 35 wherein the oligonucleotide is from a 18-mer to 26-mer.
37. Use according to claim 36 wherein the olîgonucleotide is from a 18-mer to 21-mer.
38. Use according to claim 35 wherein the oligonucleotide is an oligodeoxynucleotide selected from the group consisting of:
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.
39. Use according to claim 35 wherein the oligonucleotide is an oligodeoxynucleotide selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.
40. Use according to claim 39 wherein the oligonucleotide comprises SEQ ID NO:22.
41. Use according to claim 35 wherein the oligonucleotide is a phosphorothioate oligodeoxynucleotide or methylphosphonate oligodeoxynucleoside having a nucleotide/nucleoside sequence corresponding to any of the following:
SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15 SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 or SEQ ID NO:25.
SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15 SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 or SEQ ID NO:25.
42. Use according to claim 41 wherein the oligonucleotide comprises a phosphorothioate oligodeoxynucleotide.
43. Use according to claim 42 wherein the phosphorothioate oligodeoxynucleotide has a nucleotide sequence corresponding to SEQ ID NO:16.
44. A composition for the treatment of melanoma comprising a pharmaceutically acceptable carrier and an oligonucleotide which has a nucleotide sequence complementary to at least a portion of the mRNA
transcript of the human c-myb gene, said oligonucleotide being hybridizable to said mRNA transcript.
transcript of the human c-myb gene, said oligonucleotide being hybridizable to said mRNA transcript.
45. A composition according to claim 44 wherein the oligonucleotide is an at least 12-mer.
46. A composition according to claim 45 wherein the oligonucleotide is a methylphosphonate oligonucleoside or phosphorothioate oligonucleotide.
47. A composition according to claim 45 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the c-myb mRNA lying within about 40 nucleotides of the translation initiation codon.
48. A composition according to claim 45 wherein the oligonucleotide is an oligodeoxynucleotide having a deoxynucleotide sequence complementary to a portion of the c-myb mRNA transcript including the translation initiation codon of said transcript and/or the codon immediately downstream from the initiation codon.
49. A composition according to claim 45 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oligodeoxynucleotide.
50. A composition according to claim 49 wherein the oligonucleotide is a methylphosphonate oligonucleoside or a phosphorothioate oligonucleotide.
51. A composition according to claim 49 wherein the oligonucleotide is from a 15-mer to 30-mer.
52. A composition according to claim 51 wherein the oligonucleotide is from a 18-mer to 26-mer.
53. A composition according to claim 52 wherein the oligonucleotide is from a 18-mer to 21-mer.
54. A composition according to claim 51 wherein the oligonucleotide is an oligodeoxynucleotide selected from the group consisting of:
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.
55. A composition according to claim 51 wherein the oligonucleotide is an oligodeoxynucleotide selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.
56. A composition according to claim 55 wherein the oligonucleotide comprises SEQ ID NO:22.
57. A composition according to claim 51 wherein the oligonucleotide is a phosphorothioate oligodeoxynucleotide or methylphosphonate oligodeoxynucleoside having a nucleotide/nucleoside sequence corresponding to any of the following:
SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 5EQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24 or SEQ ID NO: 25.
SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 5EQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24 or SEQ ID NO: 25.
58. A composition according to claim 57 wherein the oligonucleotide comprises a phosphorothioate oligodeoxynucleotide.
59. A composition according to claim 58 wherein the phosphorothioate oligodeoxynucleotide has a nucleotide sequence corresponding to SEQ ID NO: 16.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US79299991A | 1991-11-15 | 1991-11-15 | |
| US792,999 | 1991-11-15 | ||
| PCT/US1992/009656 WO1993009789A1 (en) | 1991-11-15 | 1992-11-12 | Treatment of melanoma with antisense oligonucleotides to c-myb proto-oncogene |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2123611A1 true CA2123611A1 (en) | 1993-05-27 |
Family
ID=25158769
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002123611A Abandoned CA2123611A1 (en) | 1991-11-15 | 1992-11-12 | Treatment of melanoma with antisense oligonucleotides to c-myb proto-oncogene |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0667778A4 (en) |
| JP (1) | JPH07501525A (en) |
| AU (1) | AU3070992A (en) |
| CA (1) | CA2123611A1 (en) |
| WO (1) | WO1993009789A1 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5749847A (en) * | 1988-01-21 | 1998-05-12 | Massachusetts Institute Of Technology | Delivery of nucleotides into organisms by electroporation |
| US5989849A (en) * | 1991-05-09 | 1999-11-23 | Temple University Of The Commonwealth System Of Higher Education | Antisense of oligonucleotides to c-kit proto-oncogene and in vitro methods |
| US5646042A (en) * | 1992-08-26 | 1997-07-08 | Ribozyme Pharmaceuticals, Inc. | C-myb targeted ribozymes |
| US5658780A (en) | 1992-12-07 | 1997-08-19 | Ribozyme Pharmaceuticals, Inc. | Rel a targeted ribozymes |
| WO1995011301A1 (en) * | 1993-10-19 | 1995-04-27 | The Regents Of The University Of Michigan | P53-mediated apoptosis |
| US5618709A (en) * | 1994-01-14 | 1997-04-08 | University Of Pennsylvania | Antisense oligonucleotides specific for STK-1 and method for inhibiting expression of the STK-1 protein |
| US6207646B1 (en) | 1994-07-15 | 2001-03-27 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
| US5994320A (en) * | 1995-02-06 | 1999-11-30 | Regents Of The University Of Minnesota | Antisense oligonucleotides and methods for treating central nervous system tumors |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5098890A (en) * | 1988-11-07 | 1992-03-24 | Temple University-Of The Commonwealth System Of Higher Education | Antisence oligonucleotides to c-myb proto-oncogene and uses thereof |
-
1992
- 1992-11-12 WO PCT/US1992/009656 patent/WO1993009789A1/en not_active Application Discontinuation
- 1992-11-12 EP EP92924379A patent/EP0667778A4/en not_active Withdrawn
- 1992-11-12 AU AU30709/92A patent/AU3070992A/en not_active Abandoned
- 1992-11-12 JP JP5509357A patent/JPH07501525A/en active Pending
- 1992-11-12 CA CA002123611A patent/CA2123611A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| EP0667778A1 (en) | 1995-08-23 |
| WO1993009789A1 (en) | 1993-05-27 |
| AU3070992A (en) | 1993-06-15 |
| EP0667778A4 (en) | 1997-04-16 |
| JPH07501525A (en) | 1995-02-16 |
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| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |