用于造血干细胞基因治疗的逆转录病毒载体

造血干细胞是理想的血液疾患基因治疗靶细胞,具有整合功能的逆转录病毒是应用最广泛的基因转移载体。从莫洛尼小鼠白血病病毒衍生的逆转录病毒载体(MLV)对人造血干细胞转染效率低、不能长期稳定表达,已成为开展造血干细胞基因治疗的障碍。本文就提高基因转移效率、改进MLV载体特性和研究新的逆转录病毒载体等几方面的进展作一综述。     

用于造血干细胞基因治疗的逆转录病毒载体

造血干细胞是理想的血液疾患基因治疗靶细胞,具有整合功能的逆转录病毒是应用最广泛的基因转载体。从莫洛尼小鼠白病毒衍生的逆转录病毒载体（ＭＬＶ）对人造血干细胞转染效率低、不能长期稳定表达,已成为开展造血干细胞基因治疗的障碍。本文就提高基因转移效率、改进ＭＬＶ载体特性和研究新的逆转录病毒载体等几方面的进展作一综述。     

造血干细胞基因转染研究进展

原始的造血干细胞由于其全能性,能再生整个造血系统和免疫系统,是基因治疗的理想靶细胞。目前对其转基因的研究已取得较大发展,转染率明显提高。本文主要从腺病毒转染、逆转录病毒和慢病毒转染、转座子和人工染色体转染,以及目前的转染缺陷等方面进行综述,提出构建一个类型序列特异性的造血干细胞基因载体仍是长期目标。     

逆转录病毒载体在造血干细胞中介导的基因转移

造血干细胞是机体造血组织中一个具有自我更新和移植后能使造血功能重建的细胞群体,被转移有外源基因的造血干细胞可经骨髓移植的途径将外源基因引入到宿主体内,因此,可作为基因转移以及基因治疗的重要途径和方法。目前在造血干细胞中进行基因转移的方法有多种,以逆转录病毒载体介导的基因转移应用较多。它具有转移效率高、前病毒相对稳定整合和表达,宿主细胞通常不受损伤,以及有多种可供选择的逆转录病毒载体等特点,因而成为基因转移和可能用于临床基因治疗的最有效方法。本文对逆转录病毒载体在造血干细胞中进行基因转移的研究进展作一综述。     

靶向造血干细胞的基因治疗

造血干细胞以其自我复制和分化潜能的特点,成为基因治疗最理想的靶细胞之一。尽管仍然存在着诸如理想载体的选择、转染效率、生物安全性、转基因的高效表达等难题,不同程度地制约着靶向造血干细胞基因治疗的发展,但仍提供了一种全新的、有应用前景的疾病治疗手段。本文就相关的造血干细胞生物学特性、载体系统及临床应用方面作一综述。     

靶向造血干细胞的基因治疗

造血干细胞以其自我复制和分化潜能的特点，成为基因治疗最理想的靶细胞之一。尽管仍然存在着诸如理想载体的选择、转染效率、生物安全性、转基因的高效表达等难题，不同程度地制约着靶向造血干细胞基因治疗的发展，但仍提供了一种全新的、有应用前景的疾病治疗手段。本文就相关的造血干细胞生物学特性、载体系统及临床应用方面作一综述。     

多药耐药基因在基因治疗中的应用

人类多药耐药基因(mdr1)转染造血干细胞已在实验研究获得成功,现已进入Ⅰ期临床试验,用于肿瘤化疗中保护骨髓造血干细胞。mdr1与其他基因共同构建的双顺反子逆转录病毒载体转染可同时表达mdr1和其他基因,如为mdr1和其他耐药基因,则可用于联合化疗中保护骨髓造血干细胞;如为mdr1和其他治疗基因,mdr1则作为其他治疗基因的选择标志。     

基因洽疗中慢病毒载体的最新进展

基因治疗有望成为治疗遗传病、感染性疾病及肿瘤的有效手段。高效的基因转移方法为基因治疗的关键,目前应用最广的是来源于致瘤型逆转录病毒为代表的简单的逆转录病毒载体,但该类载体不能有效地转染非分裂细胞如造血干细胞及终末分化的神经细胞等。以人类免疫缺陷病毒Ⅰ型(HIV-Ⅰ)为代表的慢病毒为复杂的逆转录病毒。慢病毒载体具有可感染分裂细胞及非分裂细胞、转移基因片段容量较大、目的基因表达时间长、不易诱发宿主免疫反应等优点,已成为当前基因治疗中载体研究的热点。近年来对其基本生物学特性、载体改造及其应用研究均取得了较大进展。     

基因治疗中慢病毒载体的最新进展

基因治疗有望成为治疗遗传病、感染性疾病及肿瘤的有效手段。高效的基因转移方法为基因治疗的关键，目前应用最广的是来源于致瘤型逆转录病毒为代表的简单的逆转录病毒载体，但该类载体不能有效地转染非分裂细胞如造血干细胞及终末分化的神经细胞等。以人类免疫缺陷病毒I型（HIV－I）为代表的慢病毒为复杂的逆转录病毒。慢病毒载体具有可感染分裂细胞及非分裂细胞、转移基因片段容量较大、目的基因表达时间长、不易诱发宿主免疫反应等优点，已成为当前基因治疗中载体研究的热点。近年来对其基本生物学特性、载体改造及其应用研究均取得了较大进展。     

基因治疗中慢病毒载体的最新进展

基因治疗有望成为治疗遗传病、感染性疾病及肿瘤的有效手段.高效的基因转移方法为基因治疗的关键,目前应用最广的是来源于致瘤型逆转录病毒为代表的简单的逆转录病毒载体,但该类载体不能有效地转染非分裂细胞如造血干细胞及终末分化的神经细胞等.以人类免疫缺陷病毒Ⅰ型(HIV-Ⅰ)为代表的慢病毒为复杂的逆转录病毒.慢病毒载体具有可感染分裂细胞及非分裂细胞、转移基因片段容量较大、目的基因表达时间长、不易诱发宿主免疫反应等优点,已成为当前基因治疗中载体研究的热点.近年来对其基本生物学特性、载体改造及其应用研究均取得了较大进展.     

Alternative viral envelopes for oncoretroviruses to increase gene transfer into hematopoietic stem cells

The hematopoietic stem cell is the target for gene therapy of human blood disease. Low retroviral receptors for the commonly used vectors and quiescence of hematopoietic stem cells are believed to be major obstacles to the success of gene therapy. The development of new stem cell assays has allowed better understanding of the biology and phenotype of hematopoietic stem cells, leading to selection of highly enriched populations of hematopoietic stem cells. Quantitation of retrovirus receptors on these enriched populations of hematopoietic stem cells has resulted in the identification of subpopulations of cells expressing high levels of retrovirus receptors. New promising retrovirus envelopes are being developed. In this review, we discuss those issues that may help to resolve the problem of low gene transfer efficiency into human hematopoietic stem cells.     

Tissue-specific promoters for cancer gene therapy

Adenoviral cancer gene therapy approaches have resulted in promising recent results. Following only a decade of intense development, some of the crucial obstacles are now being overcome. Insufficient transduction has been the main limitation of earlier approaches. A new approach for increasing transduction of tumour cells is utilisation of replication-competent oncolytic agents, such as conditionally replicating adenoviruses (CRADs). The anti-tumour effect is caused by replication of the virus per se and, thus, replication must be restricted to tumour cells to protect normal tissues from damage. Tissue-specific promoters (TSPs) represent a powerful tool for decreasing the toxicity of cancer gene therapy to normal tissues and have previously been utilised for specific mutation compensation or delivery of prodrug-converting enzymes. However, TSPs can also be used for controlling crucial viral replication regulators and consequent restriction of replication to tumour cells. Initial clinical trials have demonstrated the safety and suggested efficacy for TSP-controlled CRADs as a novel approach for cancer gene therapy.     

Tissue-specific promoters for cancer gene therapy

Adenoviral cancer gene therapy approaches have resulted in promising recent results. Following only a decade of intense development, some of the crucial obstacles are now being overcome. Insufficient transduction has been the main limitation of earlier approaches. A new approach for increasing transduction of tumour cells is utilisation of replication-competent oncolytic agents, such as conditionally replicating adenoviruses (CRADs). The anti-tumour effect is caused by replication of the virus per se and, thus, replication must be restricted to tumour cells to protect normal tissues from damage. Tissue-specific promoters (TSPs) represent a powerful tool for decreasing the toxicity of cancer gene therapy to normal tissues and have previously been utilised for specific mutation compensation or delivery of prodrug-converting enzymes. However, TSPs can also be used for controlling crucial viral replication regulators and consequent restriction of replication to tumour cells. Initial clinical trials have demonstrated the safety and suggested efficacy for TSP-controlled CRADs as a novel approach for cancer gene therapy.     

Functional improvement of mutant keratin cells on addition of desmin: an alternative approach to gene therapy for dominant diseases

A major challenge to the concept of gene therapy for dominant disorders is the silencing or repairing of the mutant allele. Supplementation therapy is an alternative approach that aims to bypass the defective gene by inducing the expression of another gene, with similar function but not susceptible to the disrupting effect of the mutant one. Epidermolysis bullosa simplex (EBS) is a genetic skin fragility disorder caused by mutations in the genes for keratins K5 or K14, the intermediate filaments present in the basal cells of the epidermis. Keratin diseases are nearly all dominant in their inheritance. In cultured keratinocytes, mutant keratin renders cells more sensitive to a variety of stress stimuli such as osmotic shock, heat shock or scratch wounding. Using a 'severe' disease cell culture model system, we demonstrate reversion towards wild-type responses to stress after transfection with human desmin, an intermediate filament protein normally expressed in muscle cells. Such a supplementation therapy approach could be widely applicable to patients with related individual mutations and would avoid some of the financial obstacles to gene therapy for rare diseases.     

The silent treatment: siRNAs as small molecule drugs

As soon as RNA interference (RNAi) was found to work in mammalian cells, research quickly focused on harnessing this powerful endogenous and specific mechanism of gene silencing for human therapy. RNAi uses small RNAs, less than 30 nucleotides in length, to suppress expression of genes with complementary sequences. Two strategies can introduce small RNAs into the cytoplasm of cells, where they are active - a drug approach where double-stranded RNAs are administered in complexes designed for intracellular delivery and a gene therapy approach to express precursor RNAs from viral vectors. Phase I clinical studies have already begun to test the therapeutic potential of small RNA drugs that silence disease-related genes by RNAi. This review will discuss progress in developing and testing small RNAi-based drugs and potential obstacles.     

Intracellular trafficking of retroviral vectors: obstacles and advances

Retroviruses are efficient vehicles for delivering transgenes in vivo. Their ability to integrate into the host genome, providing a permanent imprint of their genes in the host, is a key asset for gene therapy. Furthermore, the lentivirus subset of retroviruses can infect nondividing as well as dividing cells. This expands the cell types capable of gene therapy, driving the development of lentiviral vectors. However, the precise mechanisms used by different retroviruses to efficiently deliver their genes into cell nuclei remains largely unclear. Understanding these molecular mechanisms may reveal features to improve the efficacy of current retroviral vectors. Moreover, this knowledge may expose elements pliable to other gene therapy vehicles to improve their in vivo performance and circumvent the biosafety concerns of using retroviral vectors. Therefore, the mechanisms underlying the early trafficking of retroviral vectors in host cells are reviewed here, as understood from studying the native retroviruses. Events after virus entry up to nuclear delivery of the viral cDNA are discussed. Cellular obstacles faced by these retroviral vectors and how they advance beyond these barriers is emphasized.     

Conditionally replicative adenovirus for gastrointestinal cancers

The clinical outcome of advanced gastrointestinal (GI) cancers (especially pancreatic and oesophageal cancers) is dismal, despite the advance of conventional therapeutic strategies. Cancer gene therapy is a category of new therapeutics, among which conditionally replicative adenovirus (CRAd) is one promising strategy to overcome existing obstacles of cancer gene therapy. Various CRAds have been developed for GI cancer treatment by taking advantage of the replication biology of adenovirus. Some CRAds have already been tested in clinical trials, but have fallen short of initial expectations. Concerns for clinical applicability include therapeutic potency, replication selectivity and interval end points in clinical trials. In addition, improvement of experimental animal models is needed for a deeper understanding of CRAd biology. Despite these obstacles, CRAds continue to be an exciting area of investigation with great potential for clinical utility. Further virological and oncological research will eventually lead to full realisation of the therapeutic potential of CRAds in the field of GI cancers.     

Gene, stem cell, and future therapies for orphan diseases

There are an estimated 7,000 "orphan diseases," but treatments are currently available for only about 5% of them. Recent progress in the advanced platforms of gene therapy, stem cell therapy, gene modification, and gene correction offers possibilities for new therapies and cures for rare diseases. Many rare diseases are genetic in origin, and gene therapy is being successfully applied to treat them. Human stem cell therapy, apart from bone marrow transplants, is still experimental. Genetic modification of stem cells can make stem cell-based products more effective. Autologous induced pluripotent stem (iPS) cells, when combined with new classes of artificial nucleases, have great potential in the ex vivo repair of specific mutated DNA sequences (zinc-finger proteins and transactivator-like effector nucleases). Patient-specific iPS cells can be corrected and transplanted back into the patient. Stem cells secrete paracrine factors that could become new therapeutic tools in the treatment of orphan diseases. Gene therapy and stem cell therapy with DNA repair are promising approaches to the treatment of rare, intractable diseases.     

Gene therapy for colorectal cancer

Colorectal cancer is an important public health problem worldwide. Gene therapy has therapeutic potential for patients with advanced or recurrent colorectal cancer, incurable by conventional treatments. To date, many strategies of gene therapy have been explored, including mutant gene correction, prodrug activation, immune stimulation and genetically-modified oncolytic viruses. Although the preclinical results of gene therapy for colorectal cancer have shown promise, gene therapy is still at an early stage of clinical development and has not yet shown a significant therapeutic benefit for patients. The main obstacles for introduction of gene therapy to patients are poor targeting selectivity of the vectors and inefficient gene transfer. As the science supporting tumour-selective vectors evolves, gene therapy may expand rapidly in the clinical practice of colorectal cancer treatment.     

Pertuzumab: evolving therapeutic strategies in the management of HER2-overexpressing breast cancer

Colorectal cancer is an important public health problem worldwide. Gene therapy has therapeutic potential for patients with advanced or recurrent colorectal cancer, incurable by conventional treatments. To date, many strategies of gene therapy have been explored, including mutant gene correction, prodrug activation, immune stimulation and genetically-modified oncolytic viruses. Although the preclinical results of gene therapy for colorectal cancer have shown promise, gene therapy is still at an early stage of clinical development and has not yet shown a significant therapeutic benefit for patients. The main obstacles for introduction of gene therapy to patients are poor targeting selectivity of the vectors and inefficient gene transfer. As the science supporting tumour-selective vectors evolves, gene therapy may expand rapidly in the clinical practice of colorectal cancer treatment.