The current study represents the first assessment of the efficacy and tolerance related to the administration of DNA/PEI complexes into a large mammalian brain. Despite the fact the PEI-mediated transfection exhibits toxicity in cultured mammalian cells,5,36
findings in studies in mice1,26,40
and the current study indicate that DNA/PEI complexes are well tolerated when administered into the CNS. One dog in the present study developed acute ataxia after bolus injections of DNA/PEI complexes. Most of the brain tissue in this study was homogenized for luciferase activity assays, which precluded histological analysis of the brain obtained in the ataxic dog. Note that the ataxia resolved within 24 hours. It is possible that any manipulation of the brain, such as simple insertion and removal of 10 needles or catheters may have caused mild signs of neurological deficits. All dogs receiving DNA/PEI complexes had elevated levels of mononuclear cells in the CSF after gene delivery. Injection of the adenovirus caused a more severe pleocytosis compared with naked DNA and DNA/PEI complexes, suggesting that the plasmids may be less inflammatory. We have previously shown in rodent models that adenovirus can cause acute inflammation, which is transient and does not affect the levels or duration of transgene expression.29-31,33,47-49
Alternatively, this could also be due to minor leakage of the adenoviral vectors into the CSF, which has been previously shown to cause inflammation in rodent models.8,44
In addition, it is worth noting that DNA/PEI complexes have been shown not to cause any apparent toxicity after direct administration into the urinary bladders in two patients with refractory bladder cancer.39
The dogs treated with DNA/PEI complexes were followed for 3 days in the present study, which precluded us from collecting data pertaining to long-term safety. Now that we have demonstrated the feasibility of using DNA/PEI complexes to transduce the CNS in the present study, further studies involving long-term end points are justified to establish the long-term impact and duration of transgene expression mediated by DNA/PEI complexes.
The highest levels of gene expression measured by the luciferase activity assay and by immunofluorescence were near the needle track. Peak gene expression was in this medial region where the needle tip was positioned during injection ( and ), with the majority of luciferase-positive cells near the needle track (). This is not surprising because the injected vector was most likely at high concentration near the needle track. In using the activity assay we also noted luciferase expression in the dorsal and ventral portions of brain tissue (). Scarce luciferase-positive cells were also seen in these regions of brain primarily near the ventricles ( right
). We do not expect this was due to cell migration but to diffusion of the vector into the CSF (as evidenced by rare luciferase-positive cells in the ventricles [ right
]), retrograde transport of the vector through the CNS, or both. We have previously documented that adenoviral-mediated gene transfer can be detected in the ipsilateral cerebral hemisphere relative to the injection site and that this is likely due to retrograde transport.53
Therefore, in the present study the distribution of transgene expression measured using the luciferase assay is consistent with our histological data and those provided in previous studies.
In the present study we used linear 2-kD PEI and found that in the best-case scenario (N/P ratio = 6) only 1.5% of gene transfer was achieved relative to adenovirus ( lower
). Many new formulations of PEI, however, have been developed recently, and these show much greater gene delivery efficiency and reduced toxicity in cultured cells.5,36
In addition, PEI has been conjugated to polyethylene glycol and targeting ligands such as transferrin; in these studies the stability of PEI/polyethylene glycol–transferrin/DNA complexes was increased in the blood,37
and transfection was markedly selective for tumor cells that overexpressed the transferrin receptor.24,38
Kloeckner et al.23
have recently developed EGF/PEI complexes that were up to 30 times more effective relative to unconjugated PEI in cultured cells expressing the EGF receptor. The idea of targeting DNA/PEI complexes to a cell surface receptor is appealing because of the increased efficiency of gene transfer and the ability to target specific cells for gene transfer. Such a strategy could be highly useful in the treatment of gliomas that overexpress tumor-specific receptors such as IL-13α2
and EGF receptor VIII.34,52
Thus, although our results show low levels of gene transfer with a linear 22-kD PEI by local injection, there is little doubt that improved versions of PEI and intravenous delivery would increase the efficacy of this nonviral approach.
High-level gene expression may not always be required if the expressed therapeutic protein is secretable or has a bystander effect. In this case, only transduction of a small number of cells could be sufficient to exert biological activity. For instance, intratumoral injection of naked DNA encoding IL-12
was shown to induce measurable IL-12 in the serum and tumor response in patients with metastatic melanoma.19
Administration of liposomes encoding IL-2
in patients with metastatic renal cell carcinoma demonstrated antitumor activity and safety.17
In both of these clinical studies complete tumor regression was achieved in selected patients.17,19
Furthermore, the authors of the aforementioned clinical trial in which DNA/PEI complexes were used to deliver a suicide gene into bladder carcinoma also demonstrated tumor regression.39
Finally, liposomes encoding a suicide gene have been delivered into malignant glioma and a 50% reduction in tumor volume was observed in two of eight patients.50
These clinical trials have demonstrated that gene transfer involving the use of nonviral vectors is a viable strategy that warrants further investigation. Therefore, even though DNA/PEI complexes were not as effective as adenoviral vectors, this may not prohibit clinical efficacy in the CNS.
Study of the canine models of spontaneous CNS disease will allow future investigators to address delivery, distribution, and safety challenges for gene therapy in the CNS. Unlike rodents, dogs are capable of expressing pain and distress and have brains large enough to allow us to administer doses of vector similar to those that could be used in humans. An example of an underutilized canine model is dogs with spontaneously occurring glioma. Canine glioma shares many features with the human disease such as invasive growth beyond the core tumor mass, similar genetic alterations, peritumoral edema, necrosis, and an extremely dismal prognosis.45
Additional examples of excellent canine models for disease affecting the CNS include those with epilepsy and lysosomal storage disorders. Treatment of spontaneous, rather than artificially induced, CNS disorders in canine models has the potential to accelerate translational gene therapy research and ultimately lead to increased human clinical efficacy.