Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
2000-07-13
2004-08-03
Falk, Anne-Marie (Department: 1636)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C530S332000
Reexamination Certificate
active
06770740
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of chemical cross-linkers, peptide chemistry and DNA carriers. More particularly, the invention provides surprisingly effective cross-linking peptides and peptide-DNA compositions with increased stability and reduced toxicity. Methods of using the peptide-DNA condensates in gene delivery and gene expression are also provided, optionally in combination with matrices, carriers and/or targeting agents.
2. Description of Related Art
Gene therapy is a growing field with far-reaching medical implications. Gene therapy can be used to replace specific genes, as in the correction of a heritable defect, and/or to deliver functionally active therapeutic genes into target cells. Other clinically applicable aspects of nucleic acid delivery involve the application of inhibitory nucleic acids, such as antisense constructs and/or ribozymes, to inhibit aberrant gene products, as in the treatment of cancer.
Initial efforts towards somatic gene therapy relied on indirect means of introducing genes into tissues, i.e., ex vivo gene therapy. In such embodiments, cells are removed from the body, transfected or infected with vectors carrying recombinant genes in vitro, and re-implanted into the body (“autologous cell transfer”). As an alternative, viral-mediated gene delivery is efficient, but is associated with drawbacks that limit its clinical application.
A variety of nonviral gene delivery carriers have been developed and tested as in vitro transfection agents, used to transiently express foreign DNA in cells in culture. Cationic lipids (Zhang et al., 1997), polylysine peptides (Wagner et al., 1990; Wyman et al., 1997; Morris et al., 1997) and cationic polymers such as polyethylenamine (Ogris et al., 1998; Boussif et al., 1995), bind electrostatically to the phosphate backbone of DNA to form complexes that mediate cellular uptake in culture.
Nonviral gene delivery using various carrier molecules has also been proposed for in vivo use (Wu and Wu, 1988; Wu et al., 1989; Wagner et al., 1990; Tang et al., 1996; Hara et al., 1997; Ogris et al., 1998). As opposed to their success in vitro, the attempted in vivo use of these agents to delivery DNA has revealed many complications. Notable downsides include those related to toxicity (Wolfert and Seymour, 1996), antigenicity (Stankovics et al., 1994), complement activation (Plank et al., 1996), solubility (Toncheva et al., 1998), blood compatibility (Yang and Huang, 1997), and stability (Kwoh et al., 1999). These complications relate to the size and charge of DNA carrier complexes and ultimately to the molecular characteristics of the carrier itself.
The high molecular weight (HMW) of most DNA carrier polymers increases the likelihood of activating the complement system (Plank et al., 1996), eliciting antigenicity (Stankovics et al., 1994), and being cytotoxic (Wolfert and Seymour, 1996). The size and heterogeneity of such polymers also significantly complicates regio-specific derivatization with ligands or polyethylene glycol (Wolfert et al., 1996) to arrive at optimized well-characterized DNA carriers that mediate efficient gene transfer in vivo.
In an attempt to circumvent the problems of HMW carriers, several low molecular weight (LMW) carrier peptides have been developed. Certain of these even mediate in vitro gene transfer as efficiently as their HMW counterparts (Wadhwa et al., 1997; Plank et al., 1999). These offer the advantage of controlled synthesis and defined purity, which then allows for a strategy of systematic optimization to increase expression levels and eliminate side effects.
However, when tested for in vivo efficacy, LMW carriers have been shown to lack sufficient stability to remain intact during circulation and thereby do not significantly protect DNA from premature metabolism in tissue (Kwoh et al., 1999). Recently, certain crosslinking agents have been applied to form caged DNA condensates by template polymerization, but thus far, these have not been shown to be transfection competent (Trubetskoy et al, 1998; 1999). The use of carriers with different isomeric forms is also being investigated (Laurent et al., 1999). In seeking a solution to the relative instability of LMW carriers, increased stability should not be over-emphasized to the detriment of gene transfer efficiency and/or gene expression.
Therefore, despite increasing attention in this field, the development of effective, low toxicity carriers for DNA delivery still constitutes a major challenge. The generation of low toxicity carriers with sufficient stability to mediate in vivo delivery and yet still provide efficient gene expression in target tissues would be a significant advance.
SUMMARY OF THE INVENTION
The present invention overcomes these and other drawbacks inherent in the prior art by providing a range of DNA carrier compositions for use in improved gene transfer methods. The invention particularly provides low molecular weight carriers that are minimal in size, reduce toxicity, function to condense DNA into small particles, have increased stability, mediate gene expression and, preferably, provide surprisingly effective gene expression levels.
The compositions and methods of the invention achieve high affinity binding to DNA using only low molecular weight (LMW) carriers. The invention is thus broadly based upon providing temporary, but sufficient, stability through molecular crosslinking of LMW carriers to DNA condensates.
Certain aspects of the invention exploit unpaired amines to provide effectively crosslinked peptide DNA condensates. Increasing the stability of peptide DNA condensates is thus achieved by introducing dialdehyde groups, such as glutaraldehyde, to crosslink surface amine groups on the peptides. LMW peptides cross-linked in this manner condense DNA into small condensates with improved stability, as demonstrated by increased resistance to shear stress induced fragmentation. Glutaraldehyde-crosslinked condensates are also significantly more resistant to in vitro metabolism by serum endonucleases and still mediate steady-state gene expression.
Important embodiments of the present invention concern LMW peptide DNA condensates with metabolic stability and reversibility, which provide high level gene expression. The LMW peptide portions of the DNA condensates incorporate multiple cysteine residues that allow the peptides to undergo oxidation to form interpeptide disulfide bonds while bound to DNA. Once in a target cell, the disulfide cross-links are reduced, releasing DNA for efficient gene expression. The reducing environment of the endosome is believed to mediate disulfide reduction and DNA release.
In preferred embodiments, the cross-linking peptides of the invention are prepared by replacing lysine residues with cysteine residues to provide low molecular weight DNA condensing peptides that spontaneously cross-link, after binding to DNA, by forming interpeptide disulfide bonds. The peptides thus contain multiple sulfhydryl groups designed to spontaneously polymerize and cross-link when bound to DNA. The stability of cross-linked peptide DNA condensates is dependent, at least in part, on the number of cysteines incorporated into the peptide. Disulfide bond formation in this manner decreases DNA condensates particle size, relative to control peptide DNA condensates, and prevents dissociation of peptide DNA condensates.
Importantly, the gene expression mediated by the cross-linked DNA condensates of the invention is not only maintained, but is actually increased 5 to 60-fold over uncrosslinked DNA condensates, depending on the number of cysteine residues. The cross-linking peptides caused elevated gene expression without increasing DNA uptake by cells, indicating a mechanism involving intracellular release of DNA triggered by disulfide bond reduction.
The invention provides panels and an admixtures of low molecular weight, synthetic cross-linking peptides (of generally less than twenty amino acids) that not only form small, stabilized DNA cond
Adami Roger C.
Collard Wendy T.
Kwok Kai Y.
McKenzie Donald L.
Park Youmie
Falk Anne-Marie
Qian Celine
The Regents of the University of Michigan
Williams, Morgan and Amerson
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