GMP lentiviral vectors (LV) have experienced an increase in their use as vectors of gene therapy for the treatment of inherited and acquired illnesses. This article reviews the current state in the development of these vectors, with particular attention to their large-scale production for use in clinical trials. Contrary to the oncoretroviral vectors which are made by stable producer cells clinical-grade LV can be in the majority of cases made by transient transfection using 293 and 293T cells that are grown within cell factory. However, recent advances are also prone to employ hollow fiber reactors as well as suspension cultures procedures, and the introduction of stable producer cells. As is typical in biotech, advanced downstream process protocols are devised to eliminate any unwanted contaminants that are derived from the process, like host cell DNA or plasmids as well as host cells' proteins. This article reviews the most recent large-scale processes for the production and purification of LV and provides the performance of their processes. In addition, new developments in the field that of cells with stable properties as well as their application to the development of production vehicles for clinical materials will be discussed.
With the initial marketing approval to market an AAV1 vector to treat of lipoprotein lipase deficiencies (Glybera(r)) across Europe, 1 viral vector-based gene therapy is rapidly becoming the standard therapy of acquired and rare illnesses that require a variety of viral vector systems are in use. Based on the reason for the treatment, as well as the targeted cells or tissues being treated either vector system is the most appropriate. When it comes to tissue division, or even cells with integrating vectors are necessary to ensure the manifestation of the transgene. In general retroviral vectors (in an overall sensu) are the preferred vectors since they ensure a steady transfer of transgenes that can be expressed.
In the majority of cases, two retroviral vectors have been created that are g-retroviral vectors that originate from murine Leukemia Viruses (MLV)2 as well as the lentiviral vectors (LV) that are mostly created of HIV-1.3 There was a time when a number of clinical trials based on usage of the MLV vectors proved successful, and even though these vectors are still being used however, the majority of clinical trials are toward the utilization for LV vectors. Many reasons could be cited for this change: (i) in contrast to g-retroviral vectors LVs are able to transduce cells that are not dividing as they transfer through the nucleus5 (ii) Their integration pattern differ from MLV vectors and are believed to be less dangerous in relation to the insertional mutagenesis6; as well as (iii) they are generated with high titer vectors.
This is the principal reason for a clear change from MLV to LV vectors , even though the manufacturing processes for LV vectors are not yet reached their full potential or the levels of the MLV vectors used.
LV vectors have been used successfully in clinical trials, in the first instance for the treatment of rare diseases, in particular, of primary immunodeficiencies7,8 and in neurodegenerative storage diseases.9,10 However, their application for the treatment of more frequent genetic and acquired diseases, including treatment of b-thalassemia,11 Parkinson's disease,12 and chimeric antigen receptor-based immunotherapy of cancer,13 have been assessed in clinics with exciting outcomes. This implies that the manufacturing process becomes an important issue because of the application of these new treatments for everyday use.
This review, which is built on sources that are publicly accessible provides the current status of manufacturing techniques for LV vectors. It also provides details on the advantages and drawbacks of current procedures (or strategies) and tools and also on the highest production levels that are achievable (titer, total vector volume) and concluding with a vision of what is to come.
LV Vector System(S)
The initial LV vector technology is founded upon HIV-1, an extremely well-studied human pathogen virus. Apart from HIV-1, other lentiviruses have been identified to act as vectors for gene transfers (TVs) however the majority of them have not been able to undergo clinical studies for example, HIV-2 (ref. 14) simian immunodeficiency viruses,15 or non primate lentiviruses including feline immunodeficiency virus,16 bovine immunodeficiency virus17 or caprine arthritis-encephalitis virus.18 Only equine infectious anemia virus (EIAV)-based vectors19 have been developed up to clinical use.
In the coming this review, we will concentrate on the HIV-1-based vector system of LV.
Four-plasmid systems
In the majority, it is based on safety concerns due to the pathogenicity HIV-1 in humans. Various versions of vectors for LV have been created and the 3rd generation has been commonly employed for R&D and clinical use currently. It is a four-plasmid array (Figure 1) comprised of three helper plasmids as well as a TV plasmid.
The choice of the helper GMP plasmids was dictated by the principle of the rational design of a split genome conditional packaging system described by Dull et al.21 This production system is associated with all required features necessary for safe use in clinics (use of nonoverlapping split-genome packaging constructs to maximally reducing the potential recombination events, which could lead to the generation of replication-competent lentivirus (RCL)).21 All accessory genes of HIV-1 (vif, vpr, vpu, and nef),22 present only in the very first generation of LV, have been removed because they are not necessary. Similarly, the regulatory tat gene, present in the second-generation LV, has been eliminated because its transacting function is dispensable as the U3 promoter of the 5' long terminal repeat (LTR) in the TV has been replaced by a constitutively active promoter sequence, such as cytomegalovirus (promoter)22, 23, 24 or Rous Sarcoma Virus (promoter)21 plus an optional enhancer25 or an inducible/repressible promoter sequence, such as 7tetO.26 This is commonly referred to as the pearl (lentivirus transfer vector construct containing chimeric Rous sarcoma virus (RSV)-HIV 5' LTRs) design or the PCC design ((CMV)-HIV 5'LTR).
These modifications result in the creation of an LV vector system that incorporates helper functions based upon using gag-pol (encoding for protein structures and enzymes of the virus) as well as rev (encoding for post-transcriptional regulatory) that are derived from HIV-1 as well as the env. While LV vectors are pseudo typed using different envelope glycoproteins that are heterologous, all larger-scale (clinical scale) vectors have made usage of glycoprotein from the vesicular stamatit is virus (VSV-g) envelope due to its improved stability in downstream processing as well as its wide transduction spectrum
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