U.S. patent application number 09/867693 was filed with the patent office on 2002-04-11 for lyophilizable and enhanced compacted nucleic acids.
Invention is credited to Cooper, Mark J., Costello, Maureen, Kowalczyk, Tomasz H., Pasumarthy, Murali K..
Application Number | 20020042388 09/867693 |
Document ID | / |
Family ID | 26964440 |
Filed Date | 2002-04-11 |
United States Patent
Application |
20020042388 |
Kind Code |
A1 |
Cooper, Mark J. ; et
al. |
April 11, 2002 |
Lyophilizable and enhanced compacted nucleic acids
Abstract
Counterions of polycations used to compact nucleic acids
profoundly affect shape and stability of particles formed. Shape is
associated with differential serum nuclease resistance and
colloidal stability. A surrogate for determining such properties
that is easy to measure is the turbidity parameter. Shape also
affects the suitability and efficacy of compacted nucleic acid
complexes for transfecting cells by various routes into a mammalian
body. Moreover, counterions such as acetate can protect compacted
nucleic acid complexes from adverse effects of lyophilization
Inventors: |
Cooper, Mark J.; (Moreland
Hills, OH) ; Pasumarthy, Murali K.; (Twinsburg,
OH) ; Kowalczyk, Tomasz H.; (University Heights,
OH) ; Costello, Maureen; (Margate City, NJ) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Family ID: |
26964440 |
Appl. No.: |
09/867693 |
Filed: |
May 31, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60287419 |
May 1, 2001 |
|
|
|
Current U.S.
Class: |
514/44R ;
435/6.18 |
Current CPC
Class: |
C12N 15/88 20130101;
A61K 9/0043 20130101; A61K 9/5192 20130101; A61K 48/00 20130101;
G01N 21/51 20130101; A61K 9/5146 20130101 |
Class at
Publication: |
514/44 ;
435/6 |
International
Class: |
A61K 048/00; C12Q
001/68 |
Claims
1. A method of estimating the colloidal stability of a preparation
of compacted nucleic acids, comprising the steps of: determining a
turbidity parameter of a solution of compacted nucleic acid,
wherein the turbidity parameter is defined as the slope of a
straight line obtained by plotting log of apparent absorbance of
light versus log of incident wavelength of the light, wherein said
wavelength is between about 330 nm and 420 nm; identifying the
preparation as colloidally stable if a turbidity parameter of less
than -3 is determined and identifying the preparation as
colloidally unstable if a turbidity parameter of greater than or
equal to -3 is determined.
2. A non-naturally occurring composition comprising unaggregated
nucleic acid complexes, each complex consisting essentially of a
single nucleic acid molecule and one or more polycation molecules,
said polycation molecules having a counterion selected from the
group consisting of acetate, bicarbonate, and chloride, wherein
said complex is compacted to a diameter which is less than (a)
double the theoretical diameter of a complex of said single nucleic
acid molecule and a sufficient number of polycation molecules to
provide a charge ratio of about 1:1, in the form of a condensed
sphere, or (b) 30 nm, whichever is larger.
3. The composition of claim 2 wherein the polycation molecules are
polylysine or a polylysine derivative.
4. The composition of claim 3 wherein the polylysine derivative is
polylysine peptide with a cysteine residue.
5. The composition of claim 2, said complex is compacted to a
diameter of less than 90 nm.
6. The composition of claim 2, wherein the nucleic acid complex is
compacted to a diameter less than 30 nm.
7. The composition of claim 2, wherein the nucleic acid complex is
compacted to a diameter less than 23 nm.
8. The composition of claim 2, wherein the nucleic acid complex is
compacted to a diameter not more than 12 nm.
9. The composition of claim 2 wherein said complex is compacted to
a diameter which is less than double the theoretical diameter of a
complex of said single nucleic acid and a sufficient number of
positively charged residues to provide a charge ratio of about 1:1,
in the form of a condensed sphere.
10. A method of preparing a composition according to claim 2 which
comprises mixing the nucleic acid with the polycation having
acetate as a counterion, at a salt concentration sufficient for
compaction of the complex.
11. The method of claim 10 in which the mixing is monitored to
detect, prevent or correct, the formation of aggregated or relaxed
complexes.
12. The method of claim 10 wherein the salt is NaCl.
13. The method of claim 10 wherein the nucleic acid and the
polycation are each, at the time of mixing, in a solution having a
salt concentration of 0.05 to 1.5 M.
14. The method of claim 10 in which the molar ratio of the
phosphate groups of the nucleic acid to the positively charged
groups of the polycation is in the range of 4:1 to 1:4.
15. The method of claim 10 in which the polycation is added to the
nucleic acid, while vortexing at high speed.
16. The method of claim 10 in which the nucleic acid is added to
the polycation, while vortexing at high speed.
17. The method of claim 10 wherein the mixing is monitored by a
method selected from the group consisting of electron microscopy,
light scattering, circular dichroism, and absorbance
measurement.
18. The method of claim 10 wherein the polycation molecules are
polylysine or a polylysine derivative.
19. The method of claim 18 wherein the polylysine derivative is
polylysine peptide with a cysteine residue.
20. A non-naturally occurring composition comprising unaggregated
nucleic acid complexes, each complex consisting essentially of a
single nucleic acid molecule and one or more polycation molecules,
wherein said polycation molecules have a counterion selected from
the group consisting of acetate, bicarbonate, and chloride, said
polycation molecule having a nucleic acid binding moiety through
which it is complexed to the nucleic acid, wherein said nucleic
acid molecule encodes at least one functional protein, wherein said
complex is compacted to a diameter which is less than double the
theoretical minimum diameter of a complex of said single nucleic
acid molecule and a sufficient number of polycation molecules to
provide a charge ratio of about 1:1, in the form of a condensed
sphere, or 30 nm, whichever is larger.
21. The composition of claim 20 wherein the polycation molecules
are polylysine or a polylysine derivative.
22. The composition of claim 21 wherein the polylysine derivative
is polylysine peptide with a cysteine residue.
23. The non-naturally occurring composition of claim 20 wherein
said nucleic acid molecule comprises a promoter which controls
transcription of an RNA molecule encoding the functional
protein.
24. The non-naturally occurring composition of claim 20 wherein the
protein is therapeutic.
25. The non-naturally occurring composition of claim 20 wherein the
complex is compacted to a diameter which is less than 50 nm.
26. The non-naturally occurring composition of claim 20 wherein the
complex is compacted to a diameter which is less than 30 nm.
27. The non-naturally occurring composition of claim 20 wherein the
nucleic acid complex is compacted to a diameter less than 23
nm.
28. The non-naturally occurring composition of claim 20 wherein the
nucleic acid complex is compacted to a diameter not more than 12
nm.
29. A non-naturally occurring composition comprising unaggregated
nucleic acid complexes, each complex consisting essentially of a
single double-stranded cDNA molecule and one or more polycation
molecules, said polycation molecules having a counterion selected
from the group consisting of acetate, bicarbonate, and chloride,
wherein said cDNA molecule encodes at least one functional protein,
wherein said complex is compacted to a diameter which is less than
double the theoretical minimum diameter of a complex of said single
cDNA molecule and a sufficient number of polycation molecules to
provide a charge ratio of about 1:1, in the form of a condensed
sphere, or 30 nm, whichever is larger.
30. The composition of claim 29 wherein the polycation molecules
are polylysine or a polylysine derivative.
31. The composition of claim 30 wherein the polylysine derivative
is polylysine peptide with a cysteine residue.
32. A non-naturally occurring composition comprising unaggregated
nucleic acid complexes, each complex consisting essentially of a
single nucleic acid molecule and one or more polycation molecules,
said polycation molecules having a counterion selected from the
group consisting of acetate, bicarbonate, and chloride, wherein
said nucleic acid molecule encodes at least one antisense nucleic
acid, wherein said complex is compacted to a diameter which is less
than double the theoretical minimum diameter of a complex of said
single nucleic acid molecule and a sufficient number of polycation
molecules to provide a charge ratio of about 1:1, in the form of a
condensed sphere, or 30 nm, whichever is larger.
33. The composition of claim 32 wherein the polycation molecules
are polylysine or a polylysine derivative.
34. The composition of claim 33 wherein the polylysine derivative
is polylysine peptide with a cysteine residue.
35. A non-naturally occurring composition comprising unaggregated
nucleic acid complexes, each complex consisting essentially of a
single nucleic acid molecule and one or more polycation molecules,
said polycation molecule having a counterion selected from the
group consisting of acetate, bicarbonate, and chloride, wherein
said nucleic acid molecule is an RNA molecule, wherein said complex
is compacted to a diameter which is less than double the
theoretical minimum diameter of a complex of said single nucleic
acid molecule and a sufficient number of polycation molecules to
provide a charge ratio of about 1:1, in the form of a condensed
sphere, or 30 nm, whichever is larger.
36. The composition of claim 35 wherein the polycation molecules
are polylysine or a polylysine derivative.
37. The composition of claim 36 wherein the polylysine derivative
is polylysine peptide with a cysteine residue.
38. A method of preparing a composition comprising unaggregated
nucleic acid complexes, each complex consisting essentially of a
single nucleic acid molecule and one or more polycation molecules,
said method comprising: mixing a nucleic acid molecule with a
polycation molecule at a salt concentration sufficient for
compaction of the complex to a diameter which is less than double
the theoretical minimum diameter of a complex of said single
nucleic acid molecule and a sufficient number of polycation
molecules to provide a charge ratio of about 1:1, in the form of a
condensed sphere, or 30 nm, whichever is larger, whereby
unaggregated nucleic acid complexes are formed, wherein each
complex consists essentially of a single nucleic acid molecule and
one or more polycation molecules, and wherein said polycation
molecules have a counterion selected from the group consisting of
bicarbonate and chloride.
39. The method of claim 38 wherein the polycation molecules are
polylysine or a polylysine derivative.
40. The method of claim 39 wherein the polylysine derivative is
polylysine peptide with a cysteine residue.
41. A method of preparing a composition comprising unaggregated
nucleic acid complexes, each complex consisting essentially of a
single nucleic acid molecule and one or more polycation molecules,
said method comprising: mixing a nucleic acid molecule with a
polycation molecule in a solvent to form a complex, said mixing
being performed in the absence of added salt, whereby the nucleic
acid forms soluble complexes with the polycation molecule without
forming aggregates, wherein each complex consists essentially of a
single nucleic acid molecule and one or more polycation molecules,
wherein the complexes have a diameter which is less than double the
theoretical minimum diameter of a complex of said single nucleic
acid molecule and a sufficient number of polycation molecules to
provide a charge ratio of about 1:1, in the form of a condensed
sphere, or 30 .mu.m, whichever is larger, wherein the polycation
has acetate as a counterion.
42. The method of claim 41 wherein the polycation molecules are
polylysine or a polylysine derivative.
43. The method of claim 42 wherein the polylysine derivative is
polylysine peptide with a cysteine residue.
44. A method of preparing a composition comprising unaggregated
nucleic acid complexes, each complex consisting essentially of a
single nucleic acid molecule and one or more polycation molecules,
said method comprising: mixing a nucleic acid molecule with a
polycation molecule in a solvent to form a complex, said mixing
being performed in the absence of added salt, whereby the nucleic
acid forms soluble complexes with the polycation molecule without
forming aggregates, wherein each complex consists essentially of a
single nucleic acid molecule and one or more polycation molecules,
wherein the complexes have a diameter which is less than double the
theoretical minimum diameter of a complex of said single nucleic
acid molecule and a sufficient number of polycation molecules to
provide a charge ratio of about 1:1, in the form of a condensed
sphere, or 30 nm, whichever is larger, wherein the polycation has a
counterion selected from the group consisting of bicarbonate and
chloride.
45. The method of claim 44 wherein the polycation molecules are
polylysine or a polylysine derivative.
46. The method of claim 45 wherein the polylysine derivative is
polylysine peptide with a cysteine residue.
47. Non-naturally occurring, soluble compacted complexes of a
nucleic acid and a polycation molecule made by the process of claim
10.
48. Non-naturally occurring, soluble compacted complexes of a
nucleic acid and a polycation molecule made by the process of claim
38.
49. Non-naturally occurring, soluble compacted complexes of a
nucleic acid and a polycation molecule made by the process of claim
41.
50. Non-naturally occurring, soluble compacted complexes of a
nucleic acid and a polycation made by the process of claim 44.
51. The complexes of claim 47 wherein the polycation molecules are
polylysine or a polylysine derivative.
52. The complexes of claim 51 wherein the polylysine derivative is
polylysine peptide with a cysteine residue
53. The complexes of claim 48 wherein the polycation molecules are
polylysine or a polylysine derivative.
54. The complexes of claim 53 wherein the polylysine derivative is
polylysine peptide with a cysteine residue.
55. The complexes of claim 49 wherein the polycation molecules are
polylysine or a polylysine derivative.
56. The complexes of claim 55 wherein the polylysine derivative is
polylysine peptide with a cysteine residue.
57. The complexes of claim 50 wherein the polycation molecules are
polylysine or a polylysine derivative.
58. The complexes of claim 57 wherein the polylysine derivative is
polylysine peptide with a cysteine residue.
59. A method of preventing or treating a disease or other clinical
condition in a subject which comprises: administering
intramuscularly or to the lung of the subject a prophylactically or
therapeutically effective amount of a composition comprising:
unaggregated nucleic acid complexes, each complex consisting
essentially of a single nucleic acid molecule and one or more
polycation molecules, said polycation molecule having acetate as a
counterion, wherein said complex is compacted to a diameter which
is less than (a) double the theoretical minimum diameter of a
complex of said single nucleic acid molecule and a sufficient
number of polycation molecules to provide a charge ratio of about
1:1, in the form of a condensed sphere, or (b) 30 nm, whichever is
larger, said nucleic acid being one whose integration,
hybridization or expression within target cells of said subject
prevents or treats said disease or other clinical condition.
60. The method of claim 59 wherein the step of administering is by
inhalation.
61. The method of claim 59 wherein the step of administering is by
intramuscular injection.
62. The method of claim 59 wherein the polycation molecules are
polylysine or a polylysine derivative.
63. The method of claim 62 wherein the polylysine derivative is
polylysine peptide with a cysteine residue.
64. A method of preventing or treating a disease or other clinical
condition in a subject which comprises: administering
intramuscularly or to the lung of the subject a prophylactically or
therapeutically effective amount of a composition comprising:
unaggregated nucleic acid complexes, each complex consisting
essentially of a single nucleic acid molecule and one or more
polycation molecules, said polycation molecule having a counterion
selected from the group consisting of bicarbonate and chloride,
wherein said complex is compacted to a diameter which is less than
(a) double the theoretical minimum diameter of a complex of said
single nucleic acid molecule and a sufficient number of polycation
molecules to provide a charge ratio of about 1:1, in the form of a
condensed sphere, or (b) 30 .mu.m, whichever is larger, said
nucleic acid being one whose integration, hybridization or
expression within target cells of said subject prevents or treats
said disease or other clinical condition.
65. The method of claim 64 wherein the polycation molecules are
polylysine or a polylysine derivative.
66. The method of claim 65 wherein the polylysine derivative is
polylysine peptide with a cysteine residue.
67. The method of claim 64 wherein the step of administering is by
inhalation.
68. The method of claim 64 wherein the step of administering is by
intramuscular injection.
69. The composition of claim 20 wherein said complex is compacted
to a diameter which is less than double the theoretical diameter of
a complex of said single nucleic acid and a sufficient number of
positively charged residues to provide a charge ratio of about 1:1,
in the form of a condensed sphere.
70. The composition of claim 29 wherein the nucleic acid complexes
are associated with a lipid.
71. The composition of claim 29 wherein said complex is compacted
to a diameter of less than 90 nm.
72. The composition of claim 29 wherein the nucleic acid complex is
compacted to a diameter less than 30 nm.
73. The composition of claim 29 wherein the nucleic acid complex is
compacted to a diameter less than 23 nm.
74. The composition of claim 29 wherein the nucleic acid complex is
compacted to a diameter not more than 12 nm.
75. The composition of claim 29 wherein said complex is compacted
to a diameter which is less than double the theoretical diameter of
a complex of said single nucleic acid and a sufficient number of
positively charged residues to provide a charge ratio of about 1:1,
in the form of a condensed sphere.
76. The composition of claim 32 wherein said complex is compacted
to a diameter of less than 90 nm.
77. The composition of claim 32 wherein the nucleic acid complex is
compacted to a diameter less than 30 nm.
78. The composition of claim 32 wherein the nucleic acid complex is
compacted to a diameter less than 23 nm.
79. The composition of claim 32 wherein the nucleic acid complex is
compacted to a diameter not more than 12 nm.
80. The composition of claim 32 wherein said complex is compacted
to a diameter which is less than double the theoretical diameter of
a complex of said single nucleic acid and a sufficient number of
positively charged residues to provide a charge ratio of about 1:1,
in the form of a condensed sphere.
81. The composition of claim 35 said complex is compacted to a
diameter of less than 90 nm.
82. The composition of claim 35 wherein the nucleic acid complex is
compacted to a diameter less than 30 nm.
83. The composition of claim 35 wherein the nucleic acid complex is
compacted to a diameter less than 23 nm.
84. The composition of claim 35 wherein the nucleic acid complex is
compacted to a diameter not more than 12 mn.
85. The composition of claim 35 wherein said complex is compacted
to a diameter which is less than double the theoretical diameter of
a complex of said single nucleic acid and a sufficient number of
positively charged residues to provide a charge ratio of about 1:1,
in the form of a condensed sphere.
86. The method of claim 38 wherein the salt is NaCl.
87. The method of claim 38 wherein the nucleic acid and the
polycation are each, at the time of mixing, in a solution having a
salt concentration of 0.05 to 1.5 M.
88. The method of claim 38 in which the mixing is monitored to
detect, prevent or correct, the formation of aggregated or relaxed
complexes.
89. The method of claim 38 in which the molar ratio of the
phosphate groups of the nucleic acid to the positively charged
groups of the polycation is in the range of 4:1 to 1:4.
90. The method of claim 38 in which the polycation is added to the
nucleic acid, while vortexing at high speed.
91. The method of claim 38 in which the nucleic acid is added to
the polycation, while vortexing at high speed.
92. The method of claim 38 wherein the mixing is monitored by a
method selected from the group consisting of electron microscopy,
light scattering, circular dichroism, and absorbance
measurement.
93. The method of claim 41 in which the mixing is monitored to
detect, prevent or correct, the formation of aggregated or relaxed
complexes.
94. The method of claim 41 in which the molar ratio of the
phosphate groups of the nucleic acid to the positively charged
groups of the polycation is in the range of 4:1 to 1:4.
95. The method of claim 41 in which the polycation is added to the
nucleic acid, while vortexing at high speed.
96. The method of claim 41 in which the nucleic acid is added to
the polycation, while vortexing at high speed.
97. The method of claim 41 wherein the mixing is monitored by a
method selected from the group consisting of electron microscopy,
light scattering, circular dichroism, and absorbance
measurement.
98. The method of claim 44 in which the mixing is monitored to
detect, prevent or correct, the formation of aggregated or relaxed
complexes.
99. The method of claim 44 in which the molar ratio of the
phosphate groups of the nucleic acid to the positively charged
groups of the polycation is in the range of 4:1 to 1:4.
100. The method of claim 44 in which the polycation is added to the
nucleic acid, while vortexing at high speed.
101. The method of claim 44 in which the nucleic acid is added to
the polycation, while vortexing at high speed.
102. The method of claim 44 wherein the mixing is monitored by a
method selected from the group consisting of electron microscopy,
light scattering, circular dichroism, and absorbance
measurement.
103. A non-naturally occurring composition comprising unaggregated
nucleic acid complexes, each complex consisting essentially of a
single nucleic acid molecule and one or more polycation molecules,
said polycation molecules having a counterion selected from the
group consisting of acetate, bicarbonate, and chloride.
104. The composition of claim 103 wherein the counterion is
acetate.
105. The composition of claim 2 wherein said polycation is
CK15-60P10 and the counterion is acetate, wherein CK15-60P10 is a
polyamino acid polymer of one N-terminal cysteine and 15-60 lysine
residues, wherein a molecule of polyethylene glycol having an
average molecular weight of 10 kdal is attached to the cysteine
residue.
106. The composition of claim 105 wherein the polycation molecule
comprises 30 residues of lysine.
107. The composition of claim 105 wherein the polycation molecule
comprises a targeting moiety.
108. The composition of claim 105, said complex is compacted to a
diameter of less than 90 mn.
109. The composition of claim 105, wherein the nucleic acid complex
is compacted to a diameter less than 30 nm.
110. The composition of claim 105, wherein the nucleic acid complex
is compacted to a diameter less than 23 nm.
111. The composition of claim 105, wherein the nucleic acid complex
is compacted to a diameter not more than 12 nm.
112. The composition of claim 105 wherein said complex is compacted
to a diameter which is less than double the theoretical diameter of
a complex of said single nucleic acid and a sufficient number of
positively charged residues to provide a charge ratio of about 1:1,
in the form of a condensed sphere.
113. The composition of claim 105 which is lyophilized.
114. The composition of claim 105 which is rehydrated after
lyophilization.
115. The composition of claim 105 which does not contain a
disaccharide.
116. A method of delivering polynucleotides to cells comprising:
contacting the composition of claim 114 with cells, whereby the
nucleic acid is delivered to and taken up by the cells.
117. The method of claim 1 16 wherein the composition does not
contain a disaccharide.
118. The composition of claim 20 wherein the polycation is
CK15-60P10, and the counterion is acetate, wherein CK15-60 is a
polyamino acid polymer of one N-terminal cysteine and 15-60 lysine
residues, wherein a molecule of polyethylene glycol having an
average molecular weight of 10 kdal is attached to the cysteine
residue.
119. The composition of claim 118 wherein the polycation molecule
comprises 30 residues of lysine.
120. The composition of claim 118 wherein the polycation molecule
comprises a targeting moiety.
121. The composition of claim 118 which is lyophilized.
122. The non-naturally occurring composition of claim 118 wherein
said nucleic acid molecule comprises a promoter which controls
transcription of an RNA molecule encoding the functional
protein.
123. The non-naturally occurring composition of claim 118 wherein
the protein is therapeutic.
124. The non-naturally occurring composition of claim 118 wherein
the complex is compacted to a diameter which is less than 50
nm.
125. The non-naturally occurring composition of claim 118 wherein
the complex is compacted to a diameter which is less than 30
nm.
126. The non-naturally occurring composition of claim 118 wherein
the nucleic acid complex is compacted to a diameter less than 23
nm.
127. The non-naturally occurring composition of claim 118 wherein
the nucleic acid complex is compacted to a diameter not more than
12 nm.
128. The composition of claim 118 wherein said complex is compacted
to a diameter which is less than double the theoretical diameter of
a complex of said single nucleic acid and a sufficient number of
positively charged residues to provide a charge ratio of about 1:1,
in the form of a condensed sphere.
129. The composition of claim 118 which is rehydrated after
lyophilization.
130. The composition of claim 118 which does not contain a
disaccharide.
131. A method of delivering polynucleotides to cells comprising:
contacting the composition of claim 129 with cells, wherein the
polynucleotide encodes a protein, whereby the protein is
expressed.
132. The composition of claim 29 wherein said polycation is
CK15-60P10, and said counterion is acetate, wherein CK15-60P 10 is
a polyamino acid polymer of one N-terminal cysteine and 15-60
lysine residues, wherein a molecule of polyethylene glycol having
an average molecular weight of 10 kdal is attached to the cysteine
residue.
133. The composition of claim 132 wherein the polycation molecule
comprises 30 residues of lysine.
134. The composition of claim 132 wherein the polycation molecule
comprises a targeting moiety.
135. The composition of claim 132 which is lyophilized.
136. The composition of claim 132 wherein said complex is compacted
to a diameter which is less than double the theoretical diameter of
a complex of said single nucleic acid and a sufficient number of
positively charged residues to provide a charge ratio of about 1:1,
in the form of a condensed sphere.
137. The composition of claim 132 which is rehydrated after
lyophilization.
138. The composition of claim 132 which does not contain a
disaccharide.
139. A method of delivering polynucleotides to cells comprising:
contacting the composition of claim 137 with cells, wherein the
polynucleotide encodes a protein, whereby the protein is
expressed.
140. The composition of claim 32 wherein said polycation is
CK15-60P10, and the counterion is acetate, wherein CK1 5-60P 10 is
a polyamino acid polymer of one N-terminal cysteine and 15-60
lysine residues, wherein a molecule of polyethylene glycol having
an average molecular weight of 10 kdal is attached to the cysteine
residue.
141. The composition of claim 140 wherein the polycation molecule
comprises 30 residues of lysine.
142. The composition of claim 140 wherein the polycation molecule
comprises a targeting moiety.
143. The composition of claim 140 which is lyophilized.
144. The composition of claim 140 wherein said complex is compacted
to a diameter which is less than double the theoretical diameter of
a complex of said single nucleic acid and a sufficient number of
positively charged residues to provide a charge ratio of about 1:1,
in the form of a condensed sphere.
145. The composition of claim 140 which is rehydrated after
lyophilization.
146. The composition of claim 140 which does not contain a
disaccharide.
147. A method of delivering polynucleotides to cells comprising:
contacting the compositions of claim 145 with cells, wherein the
polynucleotide encodes an antisense nucleic acid, whereby the
antisense nucleic acid is expressed.
148. The composition of claim 35 wherein said polycation is
CK15-60P10, and said counterion is acetate, wherein CK15-60P10 is a
polyamino acid polymer of one N-terminal cysteine and 15-60 lysine
residues, wherein a molecule of polyethylene glycol having an
average molecular weight of 10 kdal is attached to the cysteine
residue.
149. The composition of claim 148 wherein the polycation molecule
comprises 30 residues of lysine.
150. The composition of claim 148 wherein the polycation molecule
comprises a targeting moiety.
151. The composition of claim 148 which is lyophilized.
152. The composition of claim 148 which is lyophilized and
rehydrated.
153. The composition of claim 148 which does not contain a
disaccharide.
154. A method of delivering polynucleotides to cells comprising:
contacting the composition of claim 152 with cells, whereby the
polynucleotide is delivered to and taken up by the cells.
155. The method of claim 41, wherein said polycation is CK15-60P10,
and said counterion is acetate, wherein CK15-60P10 is a polyamino
acid polymer of one N-terminal cysteine and 15-60 lysine residues,
wherein a molecule of polyethylene glycol having an average
molecular weight of 10 kdal is attached to the cysteine
residue.
156. The method of claim 155 further comprising lyophilizing the
unaggregated nucleic acid complexes.
157. The method of claim 156 further comprising rehydrating the
lyophilized nucleic acid complexes.
158. The method of claim 155 wherein the polycation molecule
comprises 30 residues of lysine.
159. The method of claim 155 wherein the polycation molecule
comprises a targeting moiety.
160. A method of preparing a composition comprising unaggregated
nucleic acid complexes, each complex consisting essentially of a
single nucleic acid molecule and one or more polycation molecules,
said method comprising: mixing a nucleic acid molecule with a
polycation molecule at a salt concentration sufficient for
compaction of the complex to a diameter which is less than double
the theoretical minimum diameter of a complex of said single
nucleic acid molecule and a sufficient number of polycation
molecules to provide a charge ratio of about 1:1, in the form of a
condensed sphere, or 30 nm, whichever is larger, whereby
unaggregated nucleic acid complexes are formed, wherein each
complex consists essentially of a single nucleic acid molecule and
one or more polycation molecules, and wherein said polycation
molecules have a counterion selected from the group consisting of
acetate, bicarbonate and chloride.
161. The method of claim 160 wherein the counterion is acetate.
162. The method of claim 160 wherein the polycation molecules are
polylysine or a polylysine derivative.
163. The method of claim 162 wherein the polylysine derivative is
polylysine peptide with a cysteine residue.
164. Non-naturally occurring, soluble compacted complexes of a
nucleic acid and a polycation molecule made by the method of claim
160.
165. The method of claim 160 wherein the salt is NaCl.
166. The method of claim 160 wherein the nucleic acid and the
polycation are each, at the time of mixing, in a solution having a
salt concentration of 0.05 to 1.5 M.
167. The method of claim 160 in which the mixing is monitored to
detect, prevent or correct, the formation of aggregated or relaxed
complexes.
168. The method of claim 160 in which the molar ratio of the
phosphate groups of the nucleic acid to the positively charged
groups of the polycation is in the range of 4:1 to 1:4.
169. The method of claim 160 in which the polycation is added to
the nucleic acid, while vortexing at high speed.
170. The method of claim 160 in which the nucleic acid is added to
the polycation, while vortexing at high speed.
171. The method of claim 160 wherein the mixing is monitored by a
method selected from the group consisting of electron microscopy,
light scattering, circular diochroism, and absorbance
measurement.
172. The method of claim 160, wherein said polycation is CK15-60P10
and the counterion is acetate, wherein CK15-60P 10 is a polyamino
acid polymer of one N-terminal cysteine and 15-60 lysine residues,
wherein a molecule of polyethylene glycol having an average
molecular weight of 10 kdal is attached to the cysteine
residue.
173. The method of claim 172 further comprising lyophilizing the
unaggregated nucleic acid complexes.
174. The method of claim 173 further comprising rehydrating the
lyophilized nucleic acid complexes.
175. The method of claim 172 wherein the polycation molecule
comprises 30 residues of lysine.
176. The method of claim 172 wherein the polycation molecule
comprises a targeting moiety.
Description
[0001] This application claims the benefit of application Ser. Nos.
60/287,419 filed May 1, 2001 and No. 60/207,949 filed May 31, 2000,
the disclosures of which are expressly incorporated herein.
BACKGROUND OF THE INVENTION
[0002] Despite the promise of preclinical models for systemic gene
therapy to liver, lung, and other tissues, there is currently no
commercial gene therapy product on the market. The failure of most
human gene therapy clinical trials to treat metabolic disorders and
cancer has been ascribed to the relative inefficiency of viral and
non-viral gene transfer systems. Viral vectors have been used for
most gene therapy studies because of their ability to efficiently
infect cells in tissue culture. However, an enormous payload of
particles needs to be applied in an intravenous injection to
transduce cells in vivo, and toxicities of viral vectors are well
documented [1], including a recent lethal toxicity that occurred
following a portal vein injection of recombinant adenovirus [2]. In
contrast, non-viral systems are generally felt to be safe although
inefficient. There is a growing consensus that non-viral systems
will be the vector of choice for in vivo applications once gene
transfer efficiency is improved.
[0003] Several barriers restrict non-viral methods of gene
transfer, including: i) particle stability in blood and
interstitial tissues; ii) ability of the gene transfer particle to
exit capillaries and travel to parenchymal cells; iii) cell entry
via receptor-mediated endocytosis or cell fusion; iv) stability in
and escape from endosomal and lysosomal compartments; v) diffusion
rate in the cytoplasm; vi) nuclear pore transit; and vii)
"uncoating" of DNA to permit biological function in the nucleus.
For example, numerous publications have documented the failure of
non-viral methods to transfect post-mitotic, growth-arrested cells
[3-11], presumably because the intact nuclear membrane of
non-dividing cells restricts entry of naked DNA into the nucleus
via the 25 nm nuclear pore [12-13].
[0004] Thus there is a continuing need in the art for improved
formulations and methods for delivery of genes to animals and
humans. In addition, there is a need in the art for formulations
which will be stable to storage and retain biological activity.
SUMMARY OF THE INVENTION
[0005] These and other objects of the invention are provided by one
or more of the embodiments disclosed below. In one embodiment of
the invention a method of estimating the colloidal stability of a
preparation of compacted nucleic acids is provided. A turbidity
parameter of a solution of compacted nucleic acid is determined.
The turbidity parameter is defined as the slope of a straight line
obtained by plotting log of apparent absorbance of light versus log
of incident wavelength of the light. The wavelength used is between
about 330 nm and 420 nm. A preparation is identified as colloidally
stable if a turbidity parameter of less than -3 is determined. A
preparation is identified as colloidally unstable if a turbidity
parameter of greater than or equal to -3 is determined.
[0006] According to another aspect of the invention a non-naturally
occurring composition comprising unaggregated nucleic acid
complexes is provided. Each complex consists essentially of a
single nucleic acid molecule and one or more polycation molecules.
The polycation molecules have a counterion selected from the group
consisting of acetate, bicarbonate, and chloride. The complex is
compacted to a diameter which is less than (a) double the
theoretical diameter of a complex of said single nucleic acid
molecule and a sufficient number of polycation molecules to provide
a charge ratio of about 1:1, in the form of a condensed sphere, or
(b) 30 nm, whichever is larger. Optionally, the one or more
polycation molecules of the unaggregated nucleic acid complexes are
CK15-60P10, wherein acetate is used as a counterion. CK15-60P10 is
a polyamino acid polymer of one N-terminal cysteine and 15-60
lysine residues, with a molecule of polyethylene glycol having an
average molecular weight of 10 kdal attached to the cysteine
residue.
[0007] According to another aspect of the invention a method of
preparing a composition comprising unaggregated nucleic acid
complexes is provided. Each complex consists essentially of a
single nucleic acid molecule and one or more polycation molecules.
The polycation molecules have a counterion selected from the group
consisting of acetate, bicarbonate, and chloride. The complex is
compacted to a diameter which is less than (a) double the
theoretical diameter of a complex of said single nucleic acid
molecule and a sufficient number of polycation molecules to provide
a charge ratio of about 1:1, in the form of a condensed sphere, or
(b) 30 nm, whichever is larger. The nucleic acid is mixed with the
polycation having acetate, bicarbonate, or chloride as a
counterion, at a salt concentration sufficient for compaction of
the complex. Optionally, the one or more polycation molecules of
the unaggregated nucleic acid complexes are CK15-60P10, wherein
acetate is used as a counterion. CK15-60P10 is a polyamino acid
polymer of one N-terminal cysteine and 15-60 lysine residues, with
a molecule of polyethylene glycol having an average molecular
weight of 10 kdal attached to the cysteine residue.
[0008] An additional embodiment of the invention is provided as a
method of preparing a composition comprising unaggregated nucleic
acid complexes. Each complex consists essentially of a single
nucleic acid molecule and one or more polycation molecules. A
nucleic acid molecule is mixed with a polycation molecule at a salt
concentration sufficient for compaction of the complex to a
diameter which is less than double the theoretical minimum diameter
of a complex of said single nucleic acid molecule and a sufficient
number of polycation molecules to provide a charge ratio of about
1:1, in the form of a condensed sphere, or 30 nm, whichever is
larger. Unaggregated nucleic acid complexes are formed. Optionally,
the one or more polycation molecules of the unaggregated nucleic
acid complexes are CK15-60P 10, wherein acetate is used as a
counterion. CK15-60P10 is a polyamino acid polymer of one
N-terminal cysteine and 15-60 lysine residues, with a molecule of
polyethylene glycol having an average molecular weight of 10 kdal
attached to the cysteine residue.
[0009] Also provided by the present invention is a non-naturally
occurring composition comprising unaggregated nucleic acid
complexes. Each complex consists essentially of a single nucleic
acid molecule and one or more polycation molecules. The polycation
molecules have a counterion selected from the group consisting of
acetate, bicarbonate, and chloride. The nucleic acid molecule
encodes at least one functional protein. Said complex is compacted
to a diameter which is less than double the theoretical minimum
diameter of a complex of said single nucleic acid molecule and a
sufficient number of polycation molecules to provide a charge ratio
of about 1:1, in the form of a condensed sphere, or 30 nm,
whichever is larger. Optionally, the one or more polycation
molecules of the unaggregated nucleic acid complexes are
CK15-60P10, wherein acetate is used as the counterion. CK15-60P10
is a polyamino acid polymer of one N-terminal cysteine and 15-60
lysine residues, with a molecule of polyethylene glycol having an
average molecular weight of 10 kdal attached to the cysteine
residue.
[0010] Another non-naturally occurring composition comprising
unaggregated nucleic acid complexes is also provided. Each complex
consists essentially of a single double-stranded cDNA molecule and
one or more polycation molecules. Said polycation molecules have a
counterion selected from the group consisting of acetate,
bicarbonate, and chloride. The cDNA molecule encodes at least one
functional protein. The complex is compacted to a diameter which is
less than double the theoretical minimum diameter of a complex of
said single cDNA molecule and a sufficient number of polycation
molecules to provide a charge ratio of about 1:1, in the form of a
condensed sphere, or 30 nm, whichever is larger. The nucleic acid
complexes are optionally associated with a lipid. Optionally, the
one or more polycation molecules of the unaggregated nucleic acid
complexes are CK15-60P10, wherein acetate is used as the
counterion. CK15-60P10 is a polyamino acid polymer of one
N-terminal cysteine and 15-60 lysine residues, with a molecule of
polyethylene glycol having an average molecular weight of 10 kdal
attached to the cysteine residue.
[0011] Another non-naturally occurring composition comprising
unaggregated nucleic acid complexes is provided by the present
invention. Each complex consists essentially of a single nucleic
acid molecule and one or more polycation molecules. The polycation
molecules have a counterion selected from the group consisting of
acetate, bicarbonate, and chloride. The nucleic acid molecule
encodes at least one antisense nucleic acid. The complex is
compacted to a diameter which is less than double the theoretical
minimum diameter of a complex of said single nucleic acid molecule
and a sufficient number of polycation molecules to provide a charge
ratio of about 1:1, in the form of a condensed sphere, or 30 nm,
whichever is larger. Optionally, the one or more polycation
molecules of the unaggregated nucleic acid complexes are CK15-60P
10, wherein acetate is used as the counterion. CK15-60P10 is a
polyamino acid polymer of one N-terminal cysteine and 15-60 lysine
residues, with a molecule of polyethylene glycol having an average
molecular weight of 10 kdal attached to the cysteine residue.
[0012] According to another aspect of the invention a non-naturally
occurring composition comprising unaggregated nucleic acid
complexes is provided. Each complex consists essentially of a
single nucleic acid molecule and one or more polycation molecules.
The polycation molecule has a counterion selected from the group
consisting of acetate, bicarbonate, and chloride. The nucleic acid
molecule is an RNA molecule. The complex is compacted to a diameter
which is less than double the theoretical minimum diameter of a
complex of said single nucleic acid molecule and a sufficient
number of polycation molecules to provide a charge ratio of about
1:1, in the form of a condensed sphere, or 30 nm, whichever is
larger. Optionally, the one or more polycation molecules of the
unaggregated nucleic acid complexes are CK15-60P10, wherein acetate
is used as the counterion. CK15-60P10 is a polyamino acid polymer
of one N-terminal cysteine and 15-60 lysine residues with a
molecule of polyethylene glycol having an average molecular weight
of 10 kdal is attached to the cysteine residue.
[0013] Another aspect of the invention provided here is a method of
preparing a composition comprising unaggregated nucleic acid
complexes. Each complex consists essentially of a single nucleic
acid molecule and one or more polycation molecules. A nucleic acid
molecule is mixed with a polycation molecule in a solvent to form a
complex. The mixing is performed in the absence of added salt,
whereby the nucleic acid forms soluble complexes with the
polycation molecule without forming aggregates. Each complex
consists essentially of a single nucleic acid molecule and one or
more polycation molecules. The complexes have a diameter which is
less than double the theoretical minimum diameter of a complex of
the single nucleic acid molecule and a sufficient number of
polycation molecules to provide a charge ratio of about 1:1, in the
form of a condensed sphere, or 30 nm, whichever is larger. The
polycation has acetate, bicarbonate, or chloride as a counterion.
Optionally, the one or more polycation molecules of the
unaggregated nucleic acid complexes are CK15-60P10, wherein acetate
is used as the counterion. CK15-60P 10 is a polyamino acid polymer
of one N-terminal cysteine and 15-60 lysine residues with a
molecule of polyethylene glycol having an average molecular weight
of 10 kdal is attached to the cysteine residue.
[0014] Finally, the present invention provides a method of
preventing or treating a disease or other clinical condition in a
subject. A prophylactically or therapeutically effective amount of
a composition is administered intramuscularly or to the lung. The
composition comprises: unaggregated nucleic acid complexes, each
complex consisting essentially of a single nucleic acid molecule
and one or more polycation molecules, said polycation molecule
having acetate, chloride, or bicarbonate as a counterion, wherein
said complex is compacted to a diameter which is less than (a)
double the theoretical minimum diameter of a complex of said single
nucleic acid molecule and a sufficient number of polycation
molecules to provide a charge ratio of about 1:1, in the form of a
condensed sphere, or (b) 30 nm, whichever is larger. The nucleic
acid is one whose integration, hybridization or expression within
target cells of the subject prevents or treats the disease or other
clinical condition. Optionally, the one or more polycation
molecules of the unaggregated nucleic acid complexes are CK15-60P
10, wherein acetate is used as the counterion. CK15-60P10 is a
polyamino acid polymer of one N-terminal cysteine and 15-60 lysine
residues with a molecule of polyethylene glycol having an average
molecular weight of 10 kdal is attached to the cysteine
residue.
[0015] The present invention thus provides the art with improved
analytical and therapeutic techniques for delivery of DNA to cells
by providing compacted nucleic acid compositions having improved
stability and transfectability properties.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows intramuscular (IM) injection results using TFA
(trifluoroacetate) and acetate as counterions for polylysine used
to compact DNA.
[0017] FIG. 2 shows intramuscular injection results using TFA
(trifluoroacetate) as counterion for polylysine used to compact
DNA.
[0018] FIG. 3 shows intramuscular injection results using acetate
as counterions for polylysine used to compact DNA.
[0019] FIG. 4 shows intramuscular injection results using acetate
as counterions for polylysine used to compact DNA.
[0020] FIG. 5 shows a variety of parameters varying and their
effectiveness in IM injections, including size of polylysine (CK),
polyethylene glycol substitution.
[0021] FIG. 6 shows intra-tracheal instillation of 100 ug naked and
100 ug compacted DNA compared as to amount of expression in the
lung of the instilled gene (luciferase) as a function of time after
gene transfer.
[0022] FIG. 7A shows intra-tracheal instillation of naked and
compacted DNA compared as to amount of expression in the lung of
the instilled gene (luciferase) as a function of time after gene
transfer.
[0023] FIG. 7B shows plot of data above background from FIG.
7A.
[0024] FIG. 8 shows turbidity parameter plots as a function of size
of polylysine used in compaction and counterion
[0025] FIG. 9A, FIG. 9B, and FIG. 9C show a comparison of serum
stability, turbidity parameter, and sedimentation, for various
formulations of compacted nucleic acids.
[0026] FIG. 9D tabulates the results.
[0027] FIG. 10 shows the influence of counterion on the morphology
of PEG-substituted CK30 compacted DNA as shown under the electron
microscope.
[0028] FIG. 11 shows the stability of PLASmin.TM. DNA upon freezing
and lyophilization. Particles were tested with sucrose, trehalose,
or no excipient. Particles were tested with and without
polyethylene glycol, and with TFA or acetate as the counterion. DNA
stability was assessed by a low (3400 x g x 1 min) spin to pellet
aggregates, and monitoring the absorbance of DNA in the
supernatant. Stability with acetate as the counterion surpassed
other formulations in the absence of excipient.
[0029] FIG. 12 shows assessment of the turbidity parameter before
and after lyophilization using various excipients, counterions, and
with or without polyethylene glycol. Sucrose and trehalose are very
effective in maintaining the properties of the pre-lyophilization
particles. PEG-acetate similarly was effective in maintaining the
properties.
[0030] FIG. 13 shows a visualization of particles under the
electron microscope. For particles made with CK30-PEG10k acetate in
the presence of 0.5 M trehalose, the rod-like compacted particles
look identical before and after lyophilization and rehydration.
[0031] FIG. 14 shows a visualization of particles under the
electron microscope. For particles made with CK30 TFA in the
presence of 0.5M sucrose, the ellipsoidal particles of compacted
DNA look identical before and after lyophilization and
rehydration.
[0032] FIG. 15 shows the results of gene transfer experiments using
lyophilized PLASmin.TM. complexes. Luciferase enzyme was encoded by
the complexes and its activity was measured as a means of
monitoring gene transfer. While sucrose and trehalose were
effective in protecting the gene transfer activity to all
particles, particles which contained polyethylene glycol (10 kdal)
and acetate as a counterion were surprisingly stable to
lyophilization, even in the absence of cryoprotectant excipient
(disaccharide).
[0033] FIG. 16 shows a comparison of the colloidal stability of
CK30P10K and CK45P10K DNA complexes compacted using various
counterions in 0.9% NaCl. Colloidal stability is evaluated by
sedimentation and turbidity parameter.
[0034] FIG. 17 shows an electron micrograph of plasmid DNA
compacted by CK45P 10 with chloride as a counterion. Magnification
40,000. The bar shows 100 mn.
[0035] FIG. 18 shows an agarose gel electrophoresis of DNA
compacted by PEG-ylated polylysine (CK30P10K) with various
counterions. The influence of counterions on the effective net
charge of the condensed DNA can be seen by the migration of the
compacted DNA through the gel. FIG. 18 also shows the serum
stability of the CK30P10K-DNA complexes with each of the different
counterions.
[0036] FIG. 19 shows in vivo expression of luciferase plasmid
compacted by various counterion forms of PEG-ylated polylysine
(CK30P10K) after intramuscular application. Each point represents
one animal. The solid line indicates background signal of
luciferase assay. Dose was 100 .mu.g DNA.
[0037] FIG. 20 shows in vivo expression of luciferase plasmid
compacted by various forms of PEG-ylated polylysine after
intranasal application. Acetate, bicarbonate, and TFA forms of
CD30P10K and chloride form of CK45P10K were used. The acetate
formulation was prepared either in saline or water. Each point
represents one animal. The solid line indicates background signal
of luciferase assay. Dose was 100 .mu.g DNA.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The disclosures of U.S. Pat. Nos. 5,844,107, 5,877,302,
6,008,336, 6,077,835, 5,972,901, 6,200,801, and 5,972,900 and
applications Ser. Nos. 60/145,970, 09/722,340, 09/311,553 and
60/207,949 are expressly incorporated herein.
[0039] Counterions of polycations used to compact nucleic acids
profoundly affect shape of particles formed. Shape is associated
with differential serum nuclease resistance and colloidal
stability. A surrogate for determining such properties which is
easy to measure is the turbidity parameter. Moreover, shape affects
the suitability and efficacy of compacted nucleic acid complexes
for transfecting cells by various routes into a mammalian body.
[0040] The counterion used in making compacted nucleic acid
complexes also has a significant effect on the stability of the
complexes to lyophilization. Since lyophilization is a common
process to render biologicals readily transportable and storage
stable, this finding has significant ramifications. Typically,
polyamino acid polymers contain trifluoroaceate (TFA) as a
counterion. However, this counterion is far less beneficial than
acetate for purposes of lyophilization of nucleic acid polymers, as
shown below. Particles made using acetate retain their unaggregated
nature, i.e., stay in solution better, after lyophilization and
rehydration, retain their shape, and retain their gene transfer
potential.
[0041] Particles according to the present invention contain nucleic
acids, preferably a single nucleic acid molecule. The nucleic acid
may be DNA or RNA, may be double or single-stranded, may be protein
coding or anti-sense coding or non-coding. Nucleic acids also
include analogs of RNA and DNA which are modified to enhance the
resistance to degradation in vivo. A preferred analogue is a
methylphosphonate analogue of the naturally occurring
mononucleosides. More generally, the mononucleoside analogue is any
analogue whose use results in oligonucleotides which have the
advantages of (a) an improved ability to diffuse through cell
membranes and/or (b) resistance to nuclease digestion within the
body of a subject (Miller, P. S. et al., Biochemistry 20:1874-1880
(1981)). Such nucleoside analogues are well-known in the art. The
nucleic acid molecule may be an analogue of DNA or RNA. The present
invention is not limited to use of any particular DNA or RNA
analogue, provided it is capable of fulfilling its therapeutic
purpose, has adequate resistance to nucleases, and adequate
bioavailability and cell take-up. DNA or RNA may be made more
resistant to in vivo degradation by enzymes, e.g., nucleases, by
modifying internucleoside linkages (e.g., methylphosphonates or
phosphorothioates) or by incorporating modified nucleosides (e.g.,
2'-0-methylribose or 1'-alpha-anomers). The methods used for
forming the particles are as disclosed in U.S. Pat. Nos. 5,844,107,
5,877,302, 6,008,336, 6,077,835, 5,972,901, 6,200,801, and
5,972,900 and applications Ser. Nos. 60/145,970, 09/722,340,
09/311553 and 60/207,949.
[0042] Polycations according to the present invention preferably
comprise polyamino acids such as polylysine and derivatives of
polylysine. The polycation may contain from 15-60 lysine residues,
preferably in the ranges of 15-30, 30-45, or 45-60 residues.
Preferred derivatives of polylysine are CK15, CK30, CK45, which
have an additional cysteine residue attached to polylysine polymers
of length 15, 30, and 45 residues, respectively. Other amino acids
can be readily attached to polylysine without departing from the
spirit of the invention. Other polycationic amino acid polymers can
be used such as polyarginine, or copolymers of arginine and lysine.
Polymers of non-protein amino acids, such as ornithine or
citrulline, could also be used. Any pharmaceutically approved or
appropriate polycation can be used including but not limited to
protamine, histones, polycationic lipids, putrescine, spermidine,
spermine, peptides, and polypeptides. The polycation may also
contain a targeting moiety, which is typically a ligand which binds
to a receptor on a particular type of cell. The targeting ligand
may be a polyamino acid or other chemical moiety. Specificity of
interaction of the ligand and the receptor is important for
purposes of targeting.
[0043] Conditions for making compacted nucleic acid particles are
disclosed in the aforementioned patents and applications. The
conditions may include from 0-1 M salt. The preferred salt is NaCl.
Other chaotropic salts can be used as long as they are tolerated by
the animal (or cells) to which they will be administered. Suitable
agents include Sodium sulfate (Na.sub.2 SO.sub.4), Lithium sulfate
(Li.sub.2 SO.sub.4), Ammonium sulfate ((NH.sub.4).sub.2 SO.sub.4,
Potassium sulfate (K.sub.2 SO.sub.4), Magnesium sulfate
(MgSO.sub.4), Potassium phosphate (KH.sub.2 PO.sub.4), Sodium
phosphate (NaH.sub.2 PO.sub.4), Ammonium phosphate (NH.sub.4
H.sub.2 PO.sub.4), Magnesium phosphate (MgHPO.sub.4), Magnesium
chloride (Mg Cl.sub.2), Lithium chloride (LiCi), Sodium chloride
(NaCl), Potassium chloride (KCl), Cesium chloride (CaCl), Ammonium
acetate, Potassium acetate, Sodium acetate, Sodium fluoride (NaF),
Potassium fluoride (KF), Tetramethyl ammonium chloride (TMA-Cl),
Tetrabutylammonium chloride (TBA-Cl), Triethylammoniym chloride
(TEA-Cl), and Methyltriethylammonium chloride (MTEA-Cl).
[0044] If a Target Cell Binding Moiety (TBM) is used, it must bind
specifically to an accessible structure (the "receptor") of the
intended target cells. It is not necessary that it be absolutely
specific for those cells, however, it must be sufficiently specific
for the conjugate to be therapeutically effective. Preferably, its
cross-reactivity with other cells is less than 10%, more preferably
less than 5%.
[0045] There is no absolute minimum affinity which the TBM must
have for an accessible structure of the target cell, however, the
higher the affinity, the better. Preferably, the affinity is at
least 10.sup.3 liters/mole, more preferably, at least 10.sup.6
liters/mole.
[0046] The TBM may be an antibody (or a specifically binding
fragment of an antibody, such as an Fab, Fab, V.sub.M, V.sub.L or
CDR) which binds specifically to an epitope on the surface of the
target cell. Methods for raising antibodies against cells, cell
membranes, or isolated cell surface antigens are known in the art:
(a). production of immune spleen cells: immunization with soluble
antigens Hurrell, J. G. R. (1982) Monoclonal Antibodies: Techniques
and Applications. CRC Press, Boca Raton, Fla. (b). immunization
with complex antigens: membranes, whole cells and microorganisms.
Hurrell, J. G. R. (1982) Monoclonal Antibodies: Techniques and
Applications. CRC Press, Boca Raton, Fla. (c). production of
monoclonal supernatants and ascites fluids. Andrew, S. M. and
Titus, J. A. (1991). Purification of Immunoglobulin G. in Current
Protocols in Immunology (J. E. Coligan, A. M. Kruisbeek, D. H. J.
Margulies, E. M. Shevach and W. Strober, ed.) pp. A.3.9-A.3.12.
Greene Publishing Wiley-Interscience, New York. (d). production of
polyclonal antiserum in rabbit. Garvey J. S., Cremer, N. E. and
Sussdorf, D. H (eds) (1977) Methods in Immunology: A Laboratory
Text for Instruction and Research, Third Edition. W. A. Benjamin,
North Hampton, Mass. (e). production of anti-peptide antibodies by
chemical coupling of synthetic peptides to carrier proteins
Jemmerson, R., Morrow, P. I., Klinman, N. I and Patterson, Y.
(1985). Analysis of an evolutionary conserved site on mammalian
cytochrome C using synthetic peptides. Proc. Natl Acad. Sci, U.S.A.
82, 1508-1512.
[0047] The TBM may be a lectin, for which there is a cognate
carbohydrate structure on the cell surface. The target binding
moiety may be a ligand which is specifically bound by a receptor
carried by the target cells. One class of ligands of interest are
carbohydrates, especially mono- and oligosaccharides. Suitable
ligands include galactose, lactose and mannose. Another class of
ligands of interest are peptides (which here includes proteins),
such as insulin, epidermal growth factor(s), tumor necrosis factor,
prolactin, chorionic gonadotropin, FSH, LH, glucagon, lactoferrin,
transferrin, apolipoprotein E, gp120 and albumin. The following
table lists preferred target binding moieties for various classes
of target cells:
1 Target Cells Target Binding Moiety liver cells galactose Kupffer
cells mannose macrophages mannose lung Fab fragment vs. polymeric
immunoglobulin receptor (Pig R) adipose tissue insulin lymphocytes
Fab fragment vs. CD4 or gp120 enterocyte Vitamin B12 muscle insulin
fibroblasts mannose-6-phosphate nerve cells Apolipoprotein E
[0048] Use of a target binding moiety is not strictly necessary in
the case of direct injection of compacted nucleic acid complex. The
target cell in this case is passively accessible to the compacted
complex by the injection of the complex to the vicinity of the
target cell. Target binding moieties can be attached to lysine
residues, cysteine residues, or PEG using covalent or non-covalent
interactions.
[0049] It has been found that the counterion provided in
association with the polycation profoundly affects shape, and that
shape is associated with physiologically important properties for
delivery of nucleic acids. For example, trifluoroacetate (TFA)
particles form spheroids and short rods of less than about 50 nm.
Acetate leads to longer rods of 100 to 200 .mu.m. Chloride leads to
particles which are longer and skinnier than acetate particles.
Bicarbonate leads to a mixture of rods of 100-200 nm and toroids.
Any physiologically and pharmacologically acceptable counterion can
be used with the polycation. Bromine is typically supplied with
reagent grade polylysine. It is believed that bromine is inferior
to other cations as described herein, especially with respect to
physiological acceptability. Counterions can be supplied to or
substituted on polycations by means of chromatography or dialysis,
for example. For example, the polycation can be bound to an ion
exchange resin and eluted with the desired counterion. Any method
known in the art can be used for this purposed. Interestingly, it
has been found that once a particle has been compacted into a
particular shaped particle, removal and replacement of the
counterion, such as by dialysis, does not significantly alter the
shape once assumed. Thus a favorable shape can be obtained with a
particle using a non-optimum counterion for physiological purposes
and the counterion can be replaced with a superior counterion,
while retaining the shape obtained during compaction with the
original counterion. The favorable affects on nucleic acids of the
counterions may not require compaction. Thus the polycations and
counterions can be used with non-compacted nucleic acids as
well.
[0050] The behavior of these different shaped particles in gene
delivery in animals varies significantly. Acetate particles are
superior, for example, to TFA particles for delivery to muscle and
lung. Delivery to other locations in the body may also be
accomplished. These include, without limitation, administrations
which are intratracheal, by inhalation, intradermal, topical, by
eyedrops, subcutaneous, intrathecal, by enema, enteral,
intravenous, intraarterial, intralymphatic, intraperitoneal,
intrapleural, intravesicular, intraarticular, intracardiac,
intracranial, intratumor, direct to an organ, by eardrops, by
nosedrops, intraurethral, endoscopically to the upper
gastrointestinal tract, to the sigmoid, or to the colon, by
cystoscopy, by thorascope, by arthroscope, by mediastinoscopy, by
endoscopic retrograde chlolangiopancreatography, by Omaya
reservoir, by angiography including cardiac catheterization and
cerebral angiography, intrauterine, intravaginal, to the bone
marrow, to hair follicles, to the vitreous and aqueous humor, to
the sinuses, to the ureter/pelvis of the kidney, to the fallopian
tube, and to lymph nodes.
[0051] The complexes have a diameter which is less than double the
theoretical minimum diameter of a complex of the single nucleic
acid molecule and a sufficient number of polycation molecules to
provide a charge ratio of about 1:1, in the form of a condensed
sphere. For the purposes of this invention, "about 1:1" encompasses
from 1.5:1 to 1:1.5.
[0052] Turbidity parameter can be assessed by determining the
absorbance of a composition. In a preferred embodiment a Zeiss
MCS501 UV-Vis spectrometer is used. Other spectrometers as are
known in the art can be substituted. Suitable wavelengths for
collection absorbance measurements are between about 330 nm and 420
nm.
[0053] The invention is explained in particular applications in the
examples which follow.
EXAMPLES
Example 1
[0054] Resistance to serum nucleases is, among other properties, an
important feature of any effective gene therapy vector designed to
be administered systemically. Ideally, engineering this resistance
should not compromise other desirable properties of a vector, such
as its small size and colloidal stability. We have developed
reagents and methods that permit us to reproducibly compact plasmid
DNA with polylysine-polyethylene glycol (PEG) conjugates to form
small particles having defined morphology (PLASmin.TM. complexes).
Some of these formulations are stable in serum and do not aggregate
in physiologic saline. By changing components and conditions of the
compaction procedure, size and shape of the particles can be
modified. To evaluate potential correlations between serum
stability and the physical state of PLASmin.TM. complexes, we have
prepared a matrix of 24 formulations using polylysines of various
lengths and substituted with PEG to various extents. FIG. 9D.
Polylysines having exactly 15, 30, and 45 residues were obtained by
solid-phase synthesis. These polymers contained an N-terminal
cysteine residue that was used to conjugate PEG. Various mixtures
of PEG-substituted and non-substituted polylysines we re used to
obtain different PLASmin.TM. complexes. Stability of the complexes
in 75% mouse serum was tested by incubating compacted DNA at
37.degree. C. for up to 5 days and determining half-life of DNA
degradation. Simultaneously, physical characteristics of the
complexes in 150 mM NaCl were determined. Morphology was visualized
by transmission electron microscopy (FIG. 10 and FIG. 17). DNA
condensed with acetate and bicarbonate salts of CK30 polylysine
assumed forms of long (100-300 nm) and narrow (10-20 nm) rods and
relaxed toroids (.about.50-100 nm diameter, 10-20 nm width); the
TFA salt resulted in much shorter rods (<60 nm by 20-30 nm) and
small globules (20-30 nm); the chloride form of CK30 did not
compact DNA at all (FIG. 10), while CK45/chloride (FIG. 17) gave
results similar to CK30/acetate. Colloidal instability (tendency to
aggregate) was evaluated by a sedimentation assay. Additionally,
light scattering of solutions containing PLASmin.TM. complexes was
measured and expressed as a turbidity parameter (FIG. 8). We found
that all PLASmin.TM. complexes (FIG. 9A) were much more stable in
serum than naked DNA. The half-life for compacted DNA ranged from
.about.2-17 hr, while naked DNA was completely digested within a
few minutes. We also found a correlation (r.sup.2=0.77) between
half-life of degradation and colloidal instability of PLASmin.TM.
complexes: particles that tended to aggregate were more resistant
to nucleases. The tendency to aggregate also correlated with
morphology of the complexes: rod-like complexes did not aggregate;
thus, they all showed very similar serum stability, independent of
their composition (t.sub.1/2.multidot.2-5 hr). In contrast,
spherical complexes showed various extents of tendency to aggregate
depending on polylysine chain-length and PEG content. There was
little difference in serum stability between small globules and
rod-like particles. In agreement with the prediction that
aggregated particles should scatter various light wavelengths
differently than small complexes, we found a good correlation
(r.sup.2=0.88) between colloidal instability of PLASmin.TM.
complexes and turbidity of their solutions (FIG. 9B): stable
complexes had turbidity parameter around 4 to -5 (in accordance
with the Rayleigh law), while for the largest and least stable
particles this value increased to -1.3. Consequently, the turbidity
parameter also correlated with the half-life of DNA degradation in
serum (r.sup.2=0.73; FIG. 9C). Thus, we conclude that the turbidity
parameter, which is easy to determine, can be conveniently used to
preliminarily screen various formulations of compacted DNA and
predict their colloidal stability as well as serum stability.
Example 2
[0055] Effective gene transfer to lung would facilitate therapies
for pulmonary diseases, such as cystic fibrosis, and may provide a
potent means for administering mucosal vaccines. Although direct
instillation of naked DNA into mouse airways generates measurable
transgene expression, the level of expression is low, and the
duration of expression is short. We have developed reagents and
formulation methods that compact single molecules of plasmid DNA
into 20-25 nm particles (PLASmin.TM. complexes). Unlike naked DNA,
these complexes are protected from nuclease digestion and are
stable in serum. Additionally, PLASmin.TM. complexes do not
aggregate in physiologic saline and can be concentrated to over 12
mg/ml of DNA. To determine if PLASmin.TM. complexes would generate
significant levels of gene expression in lung, we instilled naked
and PLASmin.TM. complexes into the lungs of C57BL/6J mice via
direct intratracheal administration. These compacted particles
consisted of plasmid DNA and PEG-substituted polylysine polymers
consisting of 30 lysine residues. The plasmid construct encoded a
luciferase reporter gene transcriptionally controlled by a CMV
enhancer, an elongation factor 1-alpha (EF1-alpha) promoter,
EF1-alpha intron 1, the RU5 translational enhancer from HTLV I, and
an SV40 late polyadenylation signal. A DNA dose of 100 ug was
administered in 25 or 50 ul of 150 mM NaCl. At 2, 4, 5, or 12 days
following gene transfer, extracts were prepared from both lungs and
luciferase activity was measured as relative light units per mg of
protein (FIG. 6). Whereas naked DNA generated a signal of
approximately 4,000 RLU/mg on day 2 and 1,100 RLU/mg on day 4,
PLASmin.TM. complexes generated approximately 1,100,000 RLU/mg on
day 2, and 630,000 rlu/mg on day 4. Gene expression persisted for
at least 12 days after gene transfer, although at lower levels.
These compacted DNA particles produced 400-fold enhanced gene
expression compared to naked DNA on day 2, and over 1,300-fold
improved gene expression on day 4. In contrast to whole lung
extracts, less gene expression was noted in trachea, and no
expression in liver (data not shown). In dose response studies,
peak levels of transgene expression was observed using a 100 ug
dose (FIG. 7). In summary, we have determined that PLASmin.TM.
complexes effectively deliver and express transgenes in mouse lung
following direct intra-tracheal administration. In studies in
progress, the beta-galactosidase reporter gene is being utilized to
define the cell type(s) being transfected. PLASmin.TM. complexes
may provide an appropriate gene transfer method for diverse
pulmonary diseases and/or mucosal vaccines.
Example 3
[0056] Gene transfer in muscle cells following an intramuscular
injection provides a means of safe and effective vaccination, and
provides therapeutic levels of recombinant proteins, such as factor
IX, factor VIII, or alpha-1 anti-trypsin.
[0057] To optimize formulations of PLASmin.TM. DNA for
intramuscular administration, various preparation of compacted DNA
encoding the luciferase reporter gene were administered to CD2 mice
by single injection in the tibialis anterior muscle. Gene
expression was assayed at various days post gene transfer and is
presented as relative light units (RLU)/mg protein. In FIG. 1,
expression of compacted DNA formulated with the acetate salt of
CK30 polycation (complexed with PEG 10 kD) was enhanced, as
measured by luciferase activity on both days 1 and 3, compared to
other preparations of DNA formulated with the TFA salt of CK30 or
CK45. To define further the roles of counterion type, length of
polylysine, and percent substitution of polyethylene glycol (PEG),
additional experiments were conducted. Animals received IM
injections of TFA complexes consisting of either CK30 or CK45, and
PEG sizes of either 5 or 10 kD. FIG. 2) Luciferase activity was
significantly less than that observed for CK30, PEG 10 kD, acetate
complexes in FIG. 1. The enhanced gene expression of complexes
prepared using the acetate salt of CK30, PEG 10 kD, was confirmed.
(FIG. 3) In this experiment, the CK30 polycation generated better
luciferase activity than the CK45 polymer, and CK30 yielded higher
levels of luciferase activity when complexed with 10 kD rather than
5 kD PEG. The duration of gene expression produced by acetate
complexes consisting of either CK30 or CK45, both complexes with
PEG 10 kD, were next evaluated, and the results are shown in FIG.
4. In this study, the CK30 polycation gave the best level of
reporter gene activity, and the level of activity was better on day
7 than days 1 or 3. A variety of acetate complexes were tested for
gene activity as shown in FIG. 5. These formulations included CK15,
CK30, and CK45 polycations complexed with various percentages of
PEG 10 kD. A time course to 30 days was performed. Although gene
expression on days 1, 3, and 7 appeared better using CK15 compared
to CK30, the particle sizes of some CK15 complexes were larger than
30 nm or two times the theoretical diameter of a complex of said
single nucleic acid molecule and a sufficient number of polycation
molecules to provide a charge ratio of about 1:1, in the form of a
condensed sphere. For days 1, 3, 7, and 15, at least one
preparation of CK30 compacted DNA was superior to any CK45
preparation. For CK30, the 100% PEG 10 kD complexes generated
better reporter gene activity than either the 70% or 40%
substitutions. In summary, the best formulation of compacted DNA in
these studies was the acetate salt of CK30 polycation having a 100%
substitution with PEG 10 kD.
Example 4
[0058] Prior to injection, animals are anesthetized by
intraperitoneal injection with a rodent cocktail of ketamine,
xylazine, and acepromazine. A volume of 150 ul anesthetic is
administered per mouse, at a concentration of 21.5 mg/ml ketamine,
10.7 mg/ml xylazine, and 0.36 mg/ml acepromazine. The final dose is
0.32 mg ketamine, 1.6 mg xylazine, and 0.054 mg acepromazaine per
mouse.
[0059] A volume of 25 ml of each plasmid DNA formulation is
administered intratracheally to each animal using a 22-gauge
needle. A plastic catheter is placed in the trachea of the mice via
a percutaneous approach. The resulting does per animal is 300 ug,
100 ug, 30 ug, and 10 ug DNA per mouse.
[0060] After injection, animals are anesthetized by carbon dioxide
and sacrificed. The animals are bled and rinsed intra-arterially
with phosphate buffered saline. The lungs, trachea, and liver are
isolated and rinsed in the saline. Tissue samples are immediately
frozen on liquid nitrogen, and then stored at -70.degree. C.
[0061] Lung tissue is homogenized using Polytron in lysis buffer.
Protein concentration is determined. Luciferase activity of the
homogenates is determined by luciferase assay.
Example 5
[0062] The stability of PLASmin.TM. DNA upon freezing and
lyophilization was assessed. Particles were tested with sucrose,
trehalose, or no excipient. Particles were tested with and without
polyethylene glycol, and with TFA or acetate as the counterion to
the polyethylene glycol. DNA stability was assessed by a low
(3400.times. g.times.1 min) spin to pellet aggregates, and
monitoring the absorbance of DNA in the supernatant. See FIG. 11.
Stability of the complexes with acetate as the counterion surpassed
other formulations in the absence of excipient.
Example 6
[0063] The turbidity parameter is defined as the slope of a
straight line obtained by plotting log of apparent absorbance of
light versus log of incident wavelength of the light. The
wavelength used is between about 330 nm and 420 nm. A preparation
is identified as colloidally stable if a turbidity parameter of
less than -3 is determined. A preparation is identified as
colloidally unstable if a turbidity parameter of greater than or
equal to -3 is determined.
[0064] The turbidity parameter of the compacted nucleic acid
particles was assessed before and after lyophilization using
various excipients, counterions, and with or without polyethylene
glycol. See FIG. 12. Sucrose and trehalose were found to be very
effective in maintaining the properties of the pre-lyophilization
particles. PEG-acetate similarly was effective in maintaining these
properties.
Example 7
[0065] Particles were observed under the electron microscope before
and after lyophilization. See FIG. 13. Particles made with
CK30-PEG10k acetate in the presence of 0.5 M trehalose look
similarly rod-like before and after lyophilization and
rehydration.
Example 8
[0066] Particles were observed before and after lyophilization and
rehydration under the electron microscope. The ellipsoidal
particles of compacted DNA made with CK30 TFA (counterion) in the
presence of 0.5M sucrose look identical before and after
lyophilization and rehydration. See FIG. 14.
Example 9
[0067] Gene transfer experiments using lyophilized and rehydrated
PLASmin.TM. complexes were performed, comparing them to
pre-lyophilization preparations. Luciferase enzyme was encoded by
the complexes and its activity was measured as a means of
monitoring gene transfer. While sucrose and trehalose were
effective in protecting the gene transfer activity to all
particles, particles which contained polyethylene glycol (10 kdal)
and acetate as a counterion were surprisingly stable to
lyophilization, even in the absence of cryoprotectant excipient
(disaccharide). See FIG. 15.
Example 10
[0068] Polylysines having an N-terminal cysteine and exactly 30 or
45 lysine residues (CK30 or CK45, respectively) were obtained as
trifluoroacetate (TFA) salts by solid-phase synthesis. The cysteine
residue was then used to conjugate polyethylene glycol (MW 10,000)
to form PEG-ylated polylysines CK30P10K and CK45P10K. The TFA
counterion was exchanged with acetate, bicarbonate, or chloride by
gel filtration. DNA was condensed by these polylysines, dialyzed
against 0.9% NaCl, and concentrated to 1 or 4 mg/ml using
centrifugal concentrators before analysis. Plasmid DNA having 5921
bp was comprised of kanamycin resistance and luciferase genes,
elongation factor-1.alpha. promoter and first intron, CMV enhancer,
RU5 translational enhancer from HTLV I, SV40 late polyadenylation
site, and ColE1 origin of replication was used.
[0069] Colloidal stability for the DNA complexes was determined by
measuring sedimentation of condensed DNA during centrifugation
(3,400 for 1 min) and scattering of light (turbidity) in the
wavelength range of 330-415 nm. The turbidity parameter is the
slope of a straight line obtained by plotting log of apparent
absorbance (due to scattering) vs. log of incident wavelength in a
range outside the true absorption by DNA or peptides (330-415 nm).
According to the Rayleigh law, particles that are small compared to
the wavelength of light should have Turbidity Parameter of -4.
Larger particles, however, scatter light differently and have
Turbidity Parameters in the range of .about.-1 to -3. Very large
aggregates, have a Turbidity Parameter of .about.-1. We have found
that all the tested DNA formulations were colloidally stable in
normal saline (0.9% NaCl) as judged by sedimentation and turbidity
measurements. We also found that the ability of polylysines to
condense DNA depends on type of associated counterions and length
of polylysine. CK30P10k with chloride represents the extreme case
since it does not condense DNA or condenses it very poorly. (FIG.
16).
Example 11
[0070] DNA compacted by CK30P10K with various counterions was
electrophoresed through an agarose gel to examine the effect of
counterion on net charge of condensed DNA. DNA samples were loaded
directly on the gel (1.5 .mu.g) or after trypsin treatment for 40
min (0.2 .mu.g) to remove polylysine and visualize DNA integrity
and relative quantities of supercoiled, nicked, and linear plasmid
forms. DNA either migrated to the cathode (CK30/acetate,
CK30/bicarbonate, CK45/chloride), remained in the well (CK30/TFA),
or migrated to the anode (CK30/chloride). (FIG. 18). Therefore,
counterions influence effective net charge of condensed DNA as
visualized by gel electrophoresis. Acetate and bicarbonate bound to
CK30P10k and chloride bound to CK45P10k result in slightly positive
net charge, while TFA results in electrically neutral
complexes.
[0071] Serum stability was also evaluated for each of the compacted
DNA complexes. This was assessed by incubating DNA samples with 75%
mouse serum at 37.degree. C. for 2 hr, removing polylysine by
trypsinization, and evaluating DNA integrity by gel
electrophoresis. Under these conditions, properly condensed DNA is
stable, although some nicking and linearization (very little)
occurs. Naked DNA, on the other hand, is completely digested within
a few minutes (FIG. 18). We found that the ability of polylysines
to condense and protect DNA depends on type of associated
counterions and length of polylysine. CK30P10k with chloride again
represents the extreme case since it does not condense DNA or
condenses it very poorly and does not protect against
nucleases.
Example 12
[0072] Intramuscular gene delivery was assessed for each of the
counterion forms of CK30P10K. Fifty .mu.l of DNA was injected into
quadriceps of each leg of CD-1 mice (4-6 weeks old). The total dose
was 100 .mu.g. Prior to the injection, the animals were
anesthetized by intraperitoneal injection of a rodent cocktail of
Ketamine, Xylazine, and Acepromazine. One day after the injection,
the mice were terminated and entire quadrceps removed and
processed. Protein and luciferase activity were determined. (FIG.
19).
[0073] The morphology of the compacted DNA complexes appears to
have influenced their in vivo transfection efficiency. CK30/TFA
gave the lowest expression (RLU/mg protein), CK30/acetate and
CK30/bicarbonate (more relaxed structures) gave 10-100-fold higher
RLU/mg, and CK30/chloride gave the expression at the level of naked
DNA (same as or 10-fold higher than CK30/acetate, depending on
harvest day). We have found that naked DNA is more efficient than
condensed DNA and the TFA formulation is much less efficient than
other forms of condensed DNA for intramuscular gene delivery.
Example 13
[0074] Intranasal gene delivery was assessed for each of the
counterion forms of CK30P10K. Twenty five .mu.l of DNA was
administered in 5-.mu.l aliquots into nostrils of C57/BL6 mice
using an automated pipette. The total dose was 100 .mu.g. Prior to
the injection, the animals were anesthetized by intraperitoneal
injection of a rodent cocktail of Ketamine, Xylazine, and
Acepromazine. Two days after the injection, the mice were
terminated and entire lungs removed and processed. Protein and
luciferase activity were determined (FIG. 20). In intranasal
application, the acetate, bicarbonate, and TFA formulations of
condensed DNA are the most efficient among the tested formulations,
and naked DNA and CK45/chloride were much less effective. We also
found that condensed DNA administered intranasally in water is
about 10-fold less efficient than the same DNA administered in
saline.
LITERATURE CITED
[0075] 1. Cooper, M. J. (1996) Non-infectious gene transfer and
expression systems for cancer gene therapy.
[0076] 2. Semin. Oncol. 23:172-188 Weiss, R. and Nelson, D.
Washington Post, 9/29/99, page A1.
[0077] 3. Takeshita, S., Gai, D., Leclerc, G., Pickering, J. G.,
Riesssen, R., Wier, L., and Isner, J. M. (1994) Increased gene
expression after liposome-mediated arterial gene transfer
associated with intimal smooth muscle cell proliferation. J. Clin.
Invest. 93:652-661.
[0078] 4. Zabner, J., Fasbender, A. J., Moninger, T., Poellinger,
D. A., and Welsh, M. J. (1995) Cellular and molecular barriers to
gene transfer by a cationic lipid. J. Biol. Chem.
270:18997-19007.
[0079] 5. Wilke, M., Fortunati, E., van den Broek, M., Hoogeveen,
A. T., and Scholte, B. J. (1996) Efficacy of a peptide-based gene
delivery system depends on mitotic activity. Gene Ther.
3:1133-1142.
[0080] 6. Fasbender, A., Zabner, J., Zeiher, B. G., and Welsh, M.
J. (1997) A low rate of cell proliferation and reduce DNA uptake
limit cationic lipid-mediated gene transfer to primary cultures of
ciliated human airway epithelia. Gene Ther. 41173-1180.
[0081] 7. Sebestyen, M. G., Ludtke, J. J., Bassik, M. C., Zhang,
G., Budker, V., Lukhtanov, E. A., Hagstrom, J. E., and Wolff. J. A.
(1998) DNA vector chemistry: the covalent attachment of signal
peptides to plasmid DNA. Nat. Biotechnol. 16:80-85.
[0082] 8. Jiang, C., O'Connor, S. P., Fang, S. L., Wang, K. X.,
Marshall, J., Williams, J. L., Wilburn, B., Echelard, Y., and
Cheng, S. (1998) Efficiency of cationic lipid-mediated transfection
of polarized and differentiated airway epithelial cells in vitro
and in vivo.
[0083] 9. Tseng, W. C., Haselton, F. R., and Giorgio, T. D. (1999)
Mitosis enhances transgene expression of plasmid delivered by
cationic liposomes. Biochim. Biophy. Acta 1445:53-64.
[0084] 10. Mortimer, J., Tam, P., MacLachlan, I., Graham, R. W.,
Saravolac, E. G., and Joshi, P. B. (1999) Cationic lipid-mediated
transfection of cells in culture requires mitotic activity. Gene
Ther. 6:403-411.
[0085] 11. Mirzayans, R., Aubin, R., and Paterson, M. (1992)
Differential expression and stability of foreign genes introduced
into human fibroblasts by nuclear versus cytoplasmic
microinjection. Mutat. Res. 281:115-122.
[0086] 12. Dworetzky, S. I. and Feldherr, C. M. (1988)
Translocation of RNA-coated gold particles through the nuclear
pores of oocytes. J. Cell Biol. 106:575-584.
[0087] 13. Feldherr, C. M. and Akin D. (1991) Signal-mediated
nuclear transport in proliferating and growth-arrested BALB/c 3T3
cells. J. Cell Biol. 115:933-939.
* * * * *