Systematic synthesis of nonviral gene therapy vectors

Lipitoids—novel cationic lipids for cellular delivery of plasmid DNA in vitroHuang, C-Y. et al. (1998)Chem. Biol. 5, 345–354     

阳离子聚合物在非病毒基因转染中的研究进展

基因治疗为治疗先天性遗传疾病和严重后天获得性疾病提供了一条新途径.目前,基因载体分为两类:病毒载体和非病毒载体.病毒载体转染效率高,但由于某些病毒载体存在免疫原性、致癌性、宿主DNA插入整合等缺点,从而限制了它们的应用.非病毒载体具有价格低、制备简单、安全有效、无免疫原性等优点,成为基因载体研究的热点.阳离子多聚物是非病毒载体的典型代表.文中综述近年来阳离子多聚物作为基因载体的研究现状和进展,重点介绍了阳离子多聚物基因载体的分类和与DNA的相互作用和传递机制.     

N-alkylated chitosan as a potential nonviral vector for gene transfection

Alkylated chitosans (ACSs) were prepared by modifying chitosan (CS) with alkyl bromide. The self-aggregation of ACSs in acetic acid solution was characterized by fluorescence spectroscopy and dynamic light scattering method. The results indicate that introducing alkyl side chains leads to the self-aggregation of ACSs, and CS with a 99% deacetylation degree shows no aggregation due to the electrostatic repulsion. The electrophoresis experiment demonstrates that the complex between CS and DNA was formed at a charge ratio (+/-) of 1/1; ACS/DNA complexes were formed at a lower charge ratio (+/-) of 1/4. A small amount of alkylated chitosans play the same shielding role as chitosan in protecting DNA from DNase hydrolysis. Differential scanning calorimetry (DSC) and atomic force microscopy (AFM) were employed separately to investigate the thermodynamic behavior of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/CS and DPPC/ACS mixtures and the variation in topological structure of DPPC membrane induced by CS and ACS. It is shown that CS and ACS can cause the fusion of DPPC multilamellar vesicles as well as membrane destabilization. In contrast, the perturbation effect induced by ACS is more evident due to the hydrophobic interaction. CS and ACS were used to transfer plasmid-encoding CAT into C(2)C(12) cell lines. Upon elongating the alkyl side chain, the transfection efficiency is increased and levels off after the number of carbons in the side chain exceeds 8. It is proposed that the higher transfection efficiency of ACS is attributed to the increasing entry into cells facilitated by hydrophobic interactions and easier unpacking of DNA from ACS carriers due to the weakening of electrostatic attractions between DNA and ACS.     

Novel nonviral vectors for gene delivery: Synthesis and applications

We have synthesized novel cationiclipids for gene delivery bearing an ester bond betweenthe lipid moiety and the polyamine head. We have foundthat an intramolecular rearrangement occurs duringpurification of one of the products. The rearrangementled to a cyclic lipopolyamine which was active for DNAgene transfer. The formation ofcyclization products depends on the spacer foundbetween the lipid and the polyamine. The introduction ofarginine in the linker position prevents the formation ofcyclic products. Linear as well as cyclicanalogues showed high-efficiency gene transfer whentested in vitro for luciferase gene expressionas compared to dioctadecylamidoglycyl spermineor lipofectamine and also in vivo in the Lewislung carcinoma model. The introduction of arginine in thelinker position promoted increased transfectionactivity, demonstrating that a diversity of linkers,such as short peptides or glycosides, can beintroduced into cationic lipids for targeted gene transfer.     

Novel nonviral vectors for gene delivery: Synthesis and applications

Summary We have synthesized novel cationic lipids for gene delivery bearing an ester bond between the lipid moiety and the polyamine head. We have found that an intramolecular rearrangement occurs during purification of one of the products. The rearrangement led to a cyclic lipopolyamine which was active for DNA gene transfer. The formation of cyclization products depends on the spacer found between the lipid and the polyamine. The introduction of arginine in the linker position prevents the formation of cyclic products. Linear as well as cyclic analogues showed high-efficiency gene transfer when tested in vitro for luciferase gene expression as compared to dioctadecylamidoglycyl spermine or lipofectamine and also in vivo in the Lewis lung carcinoma model. The introduction of arginine in the linker position promoted increased transfection activity, demonstrating that a diversity of linkers, such as short peptides or glycosides, can be introduced into cationic lipids for targeted gene transfer.     

Bacteria as vectors for gene therapy of cancer

Anti-cancer therapy faces major challenges, particularly in terms of specificity of treatment. The ideal therapy would eradicate tumor cells selectively with minimum side effects on normal tissue. Gene or cell therapies have emerged as realistic prospects for the treatment of cancer, and involve the delivery of genetic information to a tumor to facilitate the production of therapeutic proteins. However, there is still much to be done before an efficient and safe gene medicine is achieved, primarily developing the means of targeting genes to tumors safely and efficiently. An emerging family of vectors involves bacteria of various genera. It has been shown that bacteria are naturally capable of homing to tumors when systemically administered resulting in high levels of replication locally. Furthermore, invasive species can deliver heterologous genes intra-cellularly for tumor cell expression. Here, we review the use of bacteria as vehicles for gene therapy of cancer, detailing the mechanisms of action and successes at preclinical and clinical levels.     

Adenovirus vectors with the 100K gene deleted and their potential for multiple gene therapy applications

The 100K protein has a number of critical roles vital for successful completion of the late phases of the adenovirus (Ad) life cycle. We hypothesized that the introduction of deletions within the 100K gene would allow for the production of a series of new classes of Ad vector, including one that is replication competent but blocked in the ability to carry out many late-phase Ad functions. Such a vector would have potential for several gene therapy applications, based upon its ability to increase the copy number of the transgene encoded by the vector (via genome replication) while decreasing the side effects associated with Ad late gene expression. To efficiently produce 100K-deleted Ad ([100K-]Ad) vectors, an E1- and 100K-complementing cell line (K-16) was successfully isolated. Transfection of an [E1-,100K-]Ad vector genome into the K-16 cells readily yielded high titers of the vector. After infection of noncomplementing cells, we demonstrated that [100K-]Ad vectors have a significantly decreased ability to express several Ad late genes. Additionally, if the E1 gene was present in the infected noncomplementing cells, [100K-]Ad vectors were capable of replicating their genomes to high copy number, but were significantly blocked in their ability to efficiently encapsidate the replicated genomes. Injection of an [E1-,100K-]Ad vector in vivo also correlated with significantly decreased hepatotoxicity, as well as prolonged vector persistence. In summary, the unique properties of [100K-]Ad vectors suggest that they may have utility in a variety of gene therapy applications.     

Adenoviral vectors for gene therapy

Vectors based on human adenovirus serotypes 2 (Ad2) and 5 (Ad5) of species C possess a number of features that have favored their widespread employment for gene delivery both in␣vitro and in␣vivo. However, the use of recombinant Ad2- and Ad5-based vectors for gene therapy also suffers from a number of disadvantages. These vectors possess the tropism of the parental viruses, which infect all cells that possess the appropriate surface receptors, precluding the targeting of specific cell types. Conversely, some cell types that represent important targets for gene transfer express only low levels of the cellular receptors, which lead to inefficient infection. Another major disadvantage of Ad2- and Ad5-based vectors in␣vivo is the elicitation of both an innate and an acquired immune response. Considerable attention has therefore been focused on strategies to overcome these limitations, thereby permitting the full potential of adenoviral vectors to be realized.     

Novel strains of Salmonella typhimurium as potential vectors for gene delivery

DNA vaccines are known to induce long-term antigen specific cellular responses. We tested two new strains of Salmonella typhimurium, one carrying a mutation in a SPI-2 gene and the aroC-gene and another carrying mutations in the sifA- and aroC-genes, as potential DNA vaccine delivery vehicles. We compared them with the SL7207 strain and found that the new strains were more invasive, and that they were efficient mediators of gene transfer in vitro using EGFP as reporter gene. We tested the ability of the new strains to survive within the spleen, liver and mesenteric lymph nodes and evaluated their safety in C57/BL/6J mice.     

Applications of Sleeping Beauty transposons for nonviral gene therapy

Virus-based gene therapy has advanced to clinical trials; however, this approach may result in serious adverse events including oncogenesis and the possibility of triggering fatal immune responses. Nonviral gene delivery approaches have a better safety profile, but their in vivo application has been largely limited in the past due to their inefficient delivery into cells and lack of stable chromosomal integration that is necessary for long-term therapeutic benefit. However, recent advances suggest that the use of Sleeping Beauty transposons, a novel integrating nonviral vector system, are capable of achieving long-lasting therapeutic levels of transgene expression in preclinical settings. These observations and other ongoing relevant studies may unlock the therapeutic potential of nonviral gene therapy for human diseases.     

Designing gene delivery vectors for cardiovascular gene therapy

Genetic therapy in the cardiovascular system has been proposed for a variety of diseases ranging from prevention of vein graft failure to hypertension. Such diversity in pathogenesis requires the delivery of therapeutic genes to diverse cell types in vivo for varying lengths of time if efficient clinical therapies are to be developed. Data from extensive preclinical studies have been compiled and a certain areas have seen translation into large-scale clinical trials, with some encouraging reports. It is clear that progress within a number of disease areas is limited by a lack of suitable gene delivery vector systems through which to deliver therapeutic genes to the target site in an efficient, non-toxic manner. In general, currently available systems, including non-viral systems and viral vectors such as adenovirus (Ad) or adeno-associated virus (AAV), have a propensity to transduce non-vascular tissue with greater ease than vascular cells thereby limiting their application in cardiovascular disease. This problem has led to the development and testing of improved vector systems for cardiovascular gene delivery. Traditional viral and non-viral systems are being engineered to increase their efficiency of vascular cell transduction and diminish their affinity for other cell types through manipulation of vector:cell binding and the use of cell-selective promoters. It is envisaged that future use of such technology will substantially increase the efficacy of cardiovascular gene therapy.     

Quantifying the intracellular transport of viral and nonviral gene vectors in primary neurons

Real-time confocal particle tracking (CPT) was used to compare the transport and trafficking of polyethylenimine (PEI)/DNA nanocomplexes to that of efficient adenoviruses in live primary neurons. Surprisingly, the quantitative intracellular transport properties of PEI/DNA nonviral gene vectors are similar to that of adenoviral vectors. For example, the values of individual particle/virus transport rates and the distributions of particle/virus transport modes (i.e., the percentage undergoing active, diffusive, or subdiffusive transport) largely overlapped. In addition, both PEI/DNA vectors and adenoviruses rapidly accumulated near the cell nucleus in primary neurons despite our finding that PEI/DNA move slower in neurites than in the cell body, whereas adenoviruses move with equal rates in either location. The intracellular trafficking pathways of PEI/DNA and adenoviruses, however, were substantially different. The majority of PEI/DNA trafficked through the endolysosomal pathway so as to end up in late endosomes/lysosomes (LE/Lys), whereas adenoviruses efficiently escaped endosomes. This result suggests that the sequestration of nonviral gene vectors within acidic vesicles may be a critical barrier to gene delivery to primary neurons in the central nervous system (CNS).     

Thiophenolate clusters as potential nanosized building blocks for zinc-based nanocomposite materials: synthesis and characterization

The synthesis, the spectroscopic and structural characterization of different thiophenolate-capped zinc clusters are reported and described. In particular, different reactions of 4-chlorobenzenethiol with zinc salts yield the clusters [Me₄N][Et₃NH][Zn₄(μ-S-C₆H₄-Cl)₆(S-C₆H₄-Cl)₄] (2a), [Et₃NH]₂[Zn₄(μ-S-C₆H₄-Cl)₆(S-C₆H₄-Cl)₄] (2b), and [Me₄N]₂[Zn₄(μ-S-C₆H₄-Cl)₆(S-C₆H₄-Cl)₄] (2c), and also the thiophenolate derivative [Et₃NH]₂[Zn₄(μ-S-C₆H₅)₆(S-C₆H₅)₄] (1b) was obtained.The nanosized thiophenolate-capped clusters were investigated by ¹H and ¹³C NMR, elemental analysis, and electrospray ionization (ESI) mass spectrometry. NMR experiments provided insights into the dynamic behaviour of the clusters. The thermal decomposition patterns of 2c were analyzed in air as well as in nitrogen, indicating the formation of zinc oxide and metallic zinc, respectively. The X-ray structure of 2a revealed that the cluster core consists of an adamantane-like framework analogous to those realized in many other M₄(SR)₁₀ metal complexes.     

Effective gene therapy with nonintegrating lentiviral vectors

Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency-X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.     

Latest development in viral vectors for gene therapy

Gene therapy includes the application of various viral vectors, which represent most types and families of viruses, suitable for infection of mammalian host cells. Both hereditary diseases and acquired illnesses, such as cancer, can be targeted. Because of the various properties of each viral vector, the definition of their application range depends on factors such as packaging capacity, host range, cell- or tissue-specific targeting, replication competency, genome integration and duration of transgene expression. Recent engineering of modified viral vectors has contributed to improved gene delivery efficacy.