Lentivirus is a type of retrovirus that can integrate viral genetic information into the DNA of the host cell. Lentivirus has gone through generations of engineering to become a safe robust gene delivery vehicle for gene and cell therapies, known as the lentiviral vectors (LVs). LVs demonstrate advantages over other gene delivery methods including effective infections of both dividing and non-dividing cells, long-term stable expression of the transgene, and relatively safe integration profile. Autologous hematopoietic stem cell transplant (HSCT) in combination with gene therapy has successfully treated multiple genetic blood diseases, such as sickle cell disease and severe combined immunodeficiency, in clinical trials. In this approach, the patient’s own HSCs are collected from either bone marrow or mobilized peripheral blood, genetically modified with LVs in a cell-culture plate, and transplanted back to the same patient, thereby avoiding many immune complications associated with allogeneic HSCT. The genetically modified HSCs with the therapeutic gene can self-renew and differentiate into different blood cells, therefore providing life-long therapeutic benefits for patients. The success of lentiviral gene therapy relies on several intrinsic properties of the LVs. The first property is the viral titer, the concentration of transduction units (TU) per milliliter (mL). We and others have observed that titer decreases with increasing vector length, making it difficult to produce LVs with high titers for diseases that require large transgenes. Next, infectivity or the gene transfer capacity is a measure of how well the vector can transfer its genome into HSCs at a given dose of TU/mL. Some LVs have limited gene transfer capacity that the copies of the integrated genome do not further increase with additional TU and cannot achieve the minimum copy number required for therapeutic benefits. Although the vector length is a well-known factor that affects titer and infectivity, how the vector genome limits the lentiviral lifecycle remains elusive. In Chapter 2, we compared a “well-behaved” clinical LV, EFS-ADA (4 kb), and a “poorly-performing” β-globin LV, Lenti/βAS3-FB (8.9 kb), at different steps of lentiviral lifecycle and identified several rate-limiting steps. We observed that the viral RNA (vRNA) of Lenti/βAS3-FB, but not vRNA of EFS-ADA, was heavily truncated. These truncated vRNAs failed to be reverse transcribed and subsequently cannot be integrated into the host cell genome. We also demonstrated that virion particle formation, measured by p24 ELISA, was impaired in Lenti/βAS3-FB and other reverse-oriented LVs, as they triggered certain cellular antiviral responses. Our findings uncovered two rate-limiting steps, vRNA truncation and defective virion formation, in the lentiviral lifecycle leading to low titer and infectivity. We then developed strategies to overcome these two roadblocks. In Chapter 3, we focused on overcoming cellular restriction factors (RF) in the packaging cells by conducting a targeted CRISPR Cas9 screen to knock out potential RFs. We created a new packaging cell line CRISPRed HEK293T to Disrupt Antiviral Responses (CHEDAR) by knocking out OAS1, LDLR, and PKR. In Chapter 4, we explored several methods to improve vRNA production, such as shortening the vector length, packaging with Tat, and overexpressing transcription elongation factors. The strategies described in Chapters 3 and 4 worked additively to increase titer and infectivity of different LVs, especially those with low titer or reverse-oriented transgene cassettes. In summary, the work described in this thesis elucidates the rate-limiting steps in lentiviral production and demonstrates multiple strategies to increase titer and infectivity of LVs. We hope this work help to advance the field of gene and cell therapy by improving the production technology and reducing the cost to make the therapy more effective and accessible for patients.