Transcription and Nuclear Persistence

Once DNA successfully enters the nucleus, the DNA carrier must unpack sufficiently for the desired protein to be transcribed and translated [209]. Premature release of DNA by the carrier may result in its degradation by enzymes in lysosomes and the cytoplasm [17]. Delayed unpacking, however, may prevent transcription [209]. For example, liposome unpacking prior to nuclear entry may be necessary for gene transcription, since microinjection of these gene carriers into the nucleus hindered gene expression [117]. PEI, however, did not prevent gene expression when PEI complexes were microinjected into the nucleus [117]. It is unclear at what point in the gene delivery process DNA must unpack from its vector. Schaffer et al. found that higher short-term gene expression resulted when lower-molecular-weight polylysine was used as a DNA vector [209]. Complexes using low-molecular-weight polylysine dissociated more readily from DNA than the longer polymer chains, hypothetically allowing the transcription machinery to have faster access. Whether the DNA vector unpacks the cargo DNA outside of the nucleus or enters the nucleus prior to unpacking may depend on the vector type [198] and may affect transfection outcome.

Our group [210] and others [211,212] have recently designed biodegradable gene-carrying polymers. Degradable cationic polymers condense DNA and subsequently release it at a predetermined rate, thereby increasing delivery of free DNA to or within the cell nucleus. This type of system may be especially important when vector unpacking is a rate-limiting step. Our group has recently discovered PEI/DNA complexes in the perinuclear region within minutes after they are added to cells [118]. This adds support to the hypothesis that DNA unpacking may be a major barrier to efficient transfection, at least with some cell type/gene carrier combinations.

Plasmid DNA delivered by nonviral vectors does not generally recombine with the host chromosome, limiting the danger of improper insertions [213]. However, the extrachromosomal plasmid DNA is often susceptible to intranuclear degradation over time [213], resulting in transient gene expression. Also, the transcription of genes delivered by certain viruses, such as retroviruses and adenoviruses, have been shown to be silenced through methylation [214]. The prevention of methylation with 5-azacytidine, an inhibitor of DNA methyltransferase, allowed for an extended period of gene expression [214]. The stability and persistence of the delivered gene will help determine the profile of protein production and ultimately the effectiveness of the gene therapy.

Steps taken to understand the intracellular barriers to gene delivery have led to the rational modification and improvement of carriers. Quantitative methods, such as multiple-particle tracking, to assess to the intracellular transport of gene carriers promise to add valuable insight that may ultimately lead to nonviral carriers rivaling the efficiencies seen in viral systems.

Coping with Asthma

Coping with Asthma

If you suffer with asthma, you will no doubt be familiar with the uncomfortable sensations as your bronchial tubes begin to narrow and your muscles around them start to tighten. A sticky mucus known as phlegm begins to produce and increase within your bronchial tubes and you begin to wheeze, cough and struggle to breathe.

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