Nanoparticles containing plymeric nucleic acid homologs, pharmaceutical composition and articles of manufacture containing same and methods of use thereof

Abstract

A nanoparticle capable of delivery of an encapsulated molecule into a living cell. The nanoparticle includes an encapsulation media and an isolated nucleic acid homolog sequence. The encapsulation media is primarily polymeric. The nanoparticles release the encapsulated molecule over an extended period of time. Further disclosed are pharmaceutical compositions and articles of manufacture including nanoparticles and methods of preparing and using the nanoparticles.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. patent application Ser. No. 10 / 497,783, filed Dec. 13, 2004, which is a 371 application of PCT / IL02 / 00985, filed Dec. 5, 2002, which claims the benefit of provisional application 60 / 335,837, filed Dec. 5, 2001, all of which applications are hereby incorporated by reference in their entireties.FIELD AND BACKGROUND OF THE INVENTION[0002]The present invention relates to nanoparticles containing polymeric nucleic acid homologs, pharmaceutical compositions and articles of manufacture containing same and methods of use thereof and, more particularly, to delivery of functional polymeric nuclic acid homologs to living cells.[0003]There is a long standing interest in development of new methods for gene delivery that do not require viral vectors which are typically associated with immunogenicity, oncogenesis and unknown long-term effects (Mahato et al. (1999) Adv Genet 41:95-156). Development of these new ...

AI Technical Summary

Benefits of technology

The present invention provides a system for altering the expression level of a gene within a subject. This system includes an encapsulation media made of a biodegradable polymer and an isolated nucleic acid homolog sequence encapsulated within the media. The encapsulated nucleic acid homolog sequence is designed to release over an extended period of time. The system also includes a delivery device for introducing the nanoparticles containing the isolated nucleic acid homolog sequence into a subject. The nanoparticles are capable of delivering the isolated nucleic acid homolog sequence to cells in the subject and can be used to treat medical conditions. The invention also provides a method of blocking the expression of a specific nucleic acid sequence by administering an isolated nucleic acid homolog sequence designed to specifically hybridize to the specific nucleic acid sequence. The invention also provides a pharmaceutical composition containing the encapsulated nucleic acid homolog sequence and a physiologically acceptable carrier or excipient. The invention also provides a method of blocking the expression of a specific nucleic acid sequence by administering an isolated nucleic acid homolog sequence designed to specifically hybridize to the specific nucleic acid sequence.

Problems solved by technology

Unfortunately, known nonviral gene delivery systems or “artificial viruses” typically share two inherent disadvantages.

The first disadvantage is that they are less effective than real viruses in terms of DNA delivery capacity.

The second disadvantage is that they imitate biological functions of viruses and are therefore likely to cause an immune response and / or oncogenesis.

Cationic liposomes have as inherent disadvantages low in-vivo transfection efficiency and moderate cell toxicity.

These disadvantages render them unsuitable for use in many in-situ applications, although they are often employed in tissue culture.

Further, they exhibit low in-vivo transfection efficiency, and large amounts of pDNA are required in order to achieve a biological effect.

These disadvantages render them unsuitable for use in many in-situ applications, especially if a systemic effect is required or if local delivery is impractical.

Unfortunately, development of sustained release gene delivery systems has been slow and fraught with technical difficulties (Labhasetwar et al.

Further, they exhibit low in-vivo transfection efficiency, and large amounts of pDNA are required in order to achieve a biological effect.

These disadvantages render them unsuitable for use in many in-situ applications, especially if a systemic effect is required or if local delivery is impractical.

Thus, the primary obstacle preventing development of efficient non-viral gene delivery systems remains development of sufficient transfection efficiency.

While the methods described hereinabove are theoretically recognized, their implementation has been problematic.

Furthermore, therapeutic use of ODNs has been hampered by their inherently low cellular permeability which is generally attributed to large molecular size and high charge density.

Each of these delivery strategies has inherent disadvantages as detailed hereinabove and hereinbelow.

In summary, currently available viral gene delivery vectors share, as inherent disadvantages, a number of safety related problems including, but not limited to, immunogenicity of the vectors, formation of replication-competent viruses in patients and possible presence of contaminating agents in viral vector preparations.

Additional disadvantages result from the physical limitations imposed by the virus particle.

These additional disadvantages include lack of tissue specificity, limited insert size, and difficulties in manufacturing (Romano et al.

In summary, currently available non-viral gene delivery vectors share, as inherent disadvantages, low transfection efficiency, lack of specific targeting capability, transient expression and an immune response elicited from the unmethylated CpG islands of the bacterial DNA (Romano et al.

The use of poly-1-lysin is problematic due its polydispersity and heterogenicity which contribute to low in-vivo transfection efficiency.

47(1):99-112) enhances stability against nucleases and leads to a more ideal cellular distribution, problems of efficient cellular delivery, lysosomal degradation and lack of specificity have provided serious obstacles for therapeutic use of such particles.

Despite these advantages, PNAs are currently considered to be of only limited suitability for therapeutic use because of their poor cellular incorporation (Dean (2000) Adv.

One medical problem which may be amenable to antisense therapy is restenosis after percutaneous transluminal coronary angioplasty.

However, no significant progress has been made in exploiting the PDGF / PDGF receptors system as a point of intervention for prevention of restenosis.

This lack of progress results from an emphasis on drug based, ae opposed to gene based, therapy.

Although fully phosphorothioated ODNs have increased stability and nuclease resistance, they display high affinity for various cellular proteins, resulting in nonspecific effects.

Furthermore, high concentrations of phosphorothioated oligodeoxynucleotides (PT-ODNs) inhibit DNA polymerases and RNase H, which may render them ineffective as antisense agents (Stein (2001) J. Clin. Invest. 108:641-4).

The most common problems encountered in prior art gene therapy protocols are poor efficacy and immune response of the host to the vector.

Poor efficacy may result from failure of the delivered material to enter cells, to integrate into the genome, or to be expressed at appropriate levels.

In addition, response over the course of time is often poor.

This means that readministration, which might be advantageous, is often problematic due to the abovementioned immune response.

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